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PubMed1985

p21ras. Heterogeneous localization in transformed cells.

The cellular targets for the Kirsten murine sarcoma virus (KiMSV)-transforming protein, p21ras, are unknown. Other studies have indicated that the mature form of p21 is distributed diffusely on the cytoplasmic face of the plasma membrane. However, after fixation without buffer washes, indirect immunofluorescent staining of sparse cultures revealed a particularly well preserved cellular architecture and a strikingly heterogeneous subcellular distribution of p21 in transformed normal rat kidney (NRK) cells but not in their untransformed counterparts. The transformed cells included A KiMSV-transformed NRK line. NRK cells newly transformed with KiMSV. A temperature-sensitive (ts) KiMSV-transformed NRK line. An uninfected, spontaneously transformed NRK line in which p21 was neither phosphorylated nor overproduced. In the tsKNRK line p21 was abundant at both permissive and non-permissive temperatures; however, its distribution was heterogeneous at the permissive temperature only. Observation of this array of cells indicates that the transformation-associated p21 distribution does not require overexpression of the gene, nor phosphorylation of the protein, nor the viral oncogene. Furthermore, it is reversible in the tsKNRK cells, and so appears to be highly correlated with acquisition of a transformed morphology. Accumulations of p21 occurred preferentially in subcellular locations similar to those where ruffles were observed by phase contrast microscopy and lamellar and villous extensions were observed by scanning electron microscopy (SEM). Since enhanced ruffling is a morphological correlate of transformation in a variety of cells, the distribution of p21 observed here may relate to its function as a transforming molecule.

Cell Line↗

PubMed1985

Pneumocystis carinii: growth variables and estimates in the A549 and WI-38 VA13 human cell lines.

Recent studies indicate that rat Pneumocystis carinii can be propagated in the A549 cell line, an alveolar epithelioid cell line derived from human lung carcinoma. In the present study, growth of P. carinii was compared in the A549 cell line and the WI-38 VA13 subline 2RA, an SV40 transformed derivative of the human fetal fibroblast cell line with epithelioid morphology. Similar P. carinii growth occurred in both cell lines under optimal conditions, but the WI-38 VA13 cell line was usually more sensitive to changes in the culture system. Growth of P. carinii was affected by temperature, environmental gas mixture, motion of the cultures, and source and concentration of serum additives, but not by the presence of antibodies in the medium. A technique was developed for quantitating P. carinii in the lung inoculum which permitted analysis of P. carinii growth during the first 24 hr of culture. Inverted microscope and oil immersion phase-contrast microscopy were very helpful in monitoring the organism's stages of development and viability. Thus, this culture system should be helpful in establishing standard methodology for in vitro work with P. carinii.

Ammonium Chloride↗

PubMed2003

Effect of exposure to mitomycin C on cultured tympanic membrane fibroblasts.

OBJECTIVE: Recently, attempts have been made to prolong the patency of myringotomy site with topical use of mitomycin C (MMC). It has been shown that MMC inhibits mitosis and proliferation of ocular fibroblasts, however, there are no studies of MMC's effect on tympanic membrane fibroblasts. To investigate the effects of MMC on cultured human tympanic membrane fibroblasts and understand the cellular basis of MMC for maintain myringotomy patency, cultured fibroblasts were exposed to various concentrations of MMC for periods of 5-10 min. METHODS: Effect of MMC on cultured fibroblasts was assessed by microscopic observation and cell viability test. RESULTS: Dose-, time- dependent relationship of MMC on cultured fibroblasts was revealed. There was a significant difference between the inhibition effects of MMC at concentrations of 0.4 mg/ml and control following 5 and 10 min exposure intervals. Phase-contrast microscopy showed consistency with the antiproliferative effect of MMC at higher concentration. CONCLUSIONS: Therefore, it would appear that intraoperative use of MMC could be effective in delaying the healing of the myringotomy site and extending the period of time for myringotomy patency.

Cell Death↗

PubMed1973

Use of cobalt as a mitochondrial vital stain to study cytoplasmic exchange in matings of the basidiomycete Schizophyllum commune.

Differential labeling of mates, based on the selective uptake of cobalt by mitochondria of one of the partners, has been used to determine visually by means of phase-contrast microscopy whether transfer of mitochondria occurs after hyphal fusion. Compatible and incompatible matings of the tetrapolar basidiomycete, Schizophyllum commune, were studied. Transfer was detectable in common-A, common-AB, and fully compatible matings. It was not detectable in common-B matings

Basidiomycota↗

PubMed2004

Passive stiffness of Drosophila IFM myofibrils: a novel, high accuracy measurement method.

As the smallest muscle-cell substructure that retains the intact contractile apparatus, the single myofibril is considered the optimal specimen for muscle mechanics, although its small size also poses some technical difficulties. Myofibrils from Drosophila indirect flight muscle (IFM) are particularly difficult to study because their high passive stiffness makes them hard to handle, and too resistant to stretch to produce enough elongation for the accurate measurement of sarcomere length change. In this study, we devised a novel method for accurate stiffness measurement of single relaxed myofibrils using microfabricated cantilevers and phase contrast microscopy. A special experimental protocol was developed to minimize errors, and some data analysis strategies were used to identify and exclude spurious data. Remarkably consistent results were obtained from Drosophila IFM myofibrils. This novel, high accuracy method is potentially an effective tool for detecting small passive stiffness change in muscle mutants.

Animals↗

PubMed1992

Differential inhibition of human basal keratinocyte growth to silver sulfadiazine and mafenide acetate.

The impact of topical antimicrobial agents on improving the survival of patients with major thermal injuries is significant. However, the effects of these agents on cells responsible for wound healing has only recently received attention. Fresh human basal keratinocytes were grown in serum-free modified MCDB 153 medium under standard tissue culture conditions. Cells were subsequently exposed to concentrations of silver sulfadiazine and mafenide acetate as low as 1/100 of that used clinically over a period of 5-7 days. Cellular responses documented with hemocytometer cells counts, cellular protein assays, phase-contrast microscopy, and transmission electron microscopy show only severe toxicity to mafenide acetate. Such data imply that inhibition of wound epithelialization is greater with the use of mafenide acetate than with the use of silver sulfadiazine.

Cell Count↗

PubMed1972

An evaluation of the Celloscope 401 electronic blood cell counter.

For counting erythrocytes the instrument was precise, with a mean coefficient of variation of 1.21%. Erythrocyte counts showed close agreement with results obtained on a Coulter A electronic counter of proven accuracy. When the Celloscope 401 was modified by the manufacturers to eliminate electrical interference from other laboratory equipment, satisfactory precision and accuracy for white cell counting was obtained. Using cetrimide diluent the coefficient of variation was 1.6% but when using saponin/saline diluent the coefficient of variation was 3.5%. For leucocyte counting there was close agreement between duplicate tests performed on the Celloscope 401 and the Coulter S. The instrument was capable of satisfactory precision and accuracy in platelet counting, provided that the sedimentation method was used to obtain a platelet-rich plasma. The best results were obtained if a two-step dilution was carried out with a first dilution in 10% EDTA and a second in 2.5 mM cocaine in water. Using this method the precision study indicated a coefficient of variation of 3.11%. Close agreement was obtained between platelet counts on the Celloscope 401 when compared with the results obtained either by phase-contrast microscopy or using another electronic counter. Allowing for predilution and duplicate counts on each sample, the rate of throughput was approximately 32 samples per hour. Throughout the test period, the instrument remained electronically and mechanically stable.

Blood Cell Count↗

PubMedDate not supplied

Microridges of neoplastic cervical cells. A phase-contrast study.

Fifty consecutive cervical smears representing dysplasia, carcinoma in situ or invasive cancer were examined by phase-contrast microscopy for the presence of cells with microridges. In a majority of cases, some neoplastic cells exhibited microridges. In more advanced neoplasias, the microridges were less frequent. The absence of microridges does not appear to be a characteristic of cancer per se but may be an indicator of the degree of squamous maturation of the cell.

Carcinoma, Squamous Cell↗

PubMed2005

Heterogeneity of times required for germination and outgrowth from single spores of nonproteolytic Clostridium botulinum.

Knowledge of the distribution of growth times from individual spores and quantification of this biovariability are important if predictions of growth in food are to be improved, particularly when, as for Clostridium botulinum, growth is likely to initiate from low numbers of spores. In this study we made a novel attempt to determine the distributions of times associated with the various stages of germination and subsequent growth from spores and the relationships between these stages. The time to germination (t(germ)), time to emergence (t(emerg)), and times to reach the lengths of one (t(C1)) and two (t(C2)) mature cells were quantified for individual spores of nonproteolytic C. botulinum Eklund 17B using phase-contrast microscopy and image analysis. The times to detection for wells inoculated with individual spores were recorded using a Bioscreen C automated turbidity reader and were compatible with the data obtained microscopically. The distributions of times to events during germination and subsequent growth showed considerable variability, and all stages contributed to the overall variability in the lag time. The times for germination (t(germ)), emergence (t(emerg) - t(germ)), cell maturation (t(C1) - t(emerg)), and doubling (t(C2) - t(C1)) were not found to be correlated. Consequently, it was not possible to predict the total duration of the lag phase from information for just one of the stages, such as germination. As the variability in postgermination stages is relatively large, the first spore to germinate will not necessarily be the first spore to produce actively dividing cells and start neurotoxin production. This information can make a substantial contribution to improved predictive modeling and better quantitative microbiological risk assessment.

Bacteriological Techniques↗

PubMed2002

La(3+) and Gd(3+) induce shape change of giant unilamellar vesicles of phosphatidylcholine.

Lanthanides such as La(3+) and Gd(3+) are well known to have large effects on the function of membrane proteins such as mechanosensitive ionic channels and voltage-gated sodium channels, and also on the structure of phospholipid membranes. In this report, we have investigated effects of La(3+) and Gd(3+) on the shape of giant unilamellar vesicle (GUV) of dioleoylphosphatidylcholine (DOPC-GUV) and GUV of DOPC/cholesterol by the phase-contrast microscopy. The addition of 10-100 microM La(3+) (or Gd(3+)) through a 10-microm diameter micropipette near the DOPC-GUV (or DOPC/cholesterol-GUV) triggered several kinds of shape changes. We have found that a very low concentration (10 microM) of La(3+) (or Gd(3+)) induced a shape change of GUV such as the discocyte via stomatocyte to inside budded shape transformation, the two-spheres connected by a neck to prolate transformation, and the pearl on a string to cylinder (or tube) transformation. To understand the effect of these lanthanides on the shape of the GUV, we have also investigated phase transitions of 30 microM dipalmitoylphosphatidylcholine-multilamellar vesicle (DPPC-MLV) by the ultra-sensitive differential scanning calorimetry (DSC). The chain-melting phase transition temperature and the L(beta') to P(beta') phase transition temperature of DPPC-MLV increased with an increase in La(3+) concentration. This result indicates that the lateral compression pressure of the membrane increases with an increase in La(3+) concentration. Thereby, the interaction of La(3+) (or Gd(3+)) on the external monolayer membrane of the GUV induces a decrease in its area (A(ex)), whereas the area of the internal monolayer membrane (A(in)) keeps constant. Therefore, the shape changes of the GUV induced by these lanthanides can be explained reasonably by the decrease in the area difference between two monolayers (DeltaA=A(ex)-A(in)).

Calorimetry, Differential Scanning↗

PubMed1986

Quantitation and morphological characterization of rapid axon and dendritic growth from single cerebral hemispheric neurons in hydrated collagen lattice culture.

Quantitative and qualitative data are reported for single cerebral hemispheric neurons in a 3-dimensional hydrated collagen lattice (HCL) culture system. Individual neurons not in contact with other cells or cell processes, including synapses, rapidly displayed two morphologically distinct classes of processes that could be traced from origin to termination: long thin processes interpreted as being axons, and shorter tapering and sometimes branched processes interpreted as being dendrites. Axons and dendrites of single neurons that had at least one process longer than the cell body were measured on each of 3 days after plating using an image analysis system coupled to a phase-contrast microscope and a microcomputer. Mean lengths of axons and dendrites alone or combined as total new growth per neuron, increased 3- to 5-fold and were as high as 745, 694 and 1226 microns respectively after 3 days in HCL, although some individual axons measured over 1500 microns. Other indices of neuron growth and differentiation increased 1- to 5-fold including the number of primary processes, branch points, segments and growth cones. Phase-contrast microscopy, staining with Nissl and silver, and scanning and transmission electron microscopy demonstrated many single multipolar and other neurons with axons, dendrites and well-differentiated properties. The data show that individual central nervous system neurons have an inherent capacity to quickly express characteristic differentiated features and also to grow rapidly in HCL.

Animals↗

PubMed2004

Effect of high pressure gaseous carbon dioxide on the germination of bacterial spores.

Effect of high pressure gaseous carbon dioxide treatment (HGCT) at 6.5 MPa, 35 degrees C on the germination of bacterial spores was investigated. Germination of bacterial spores was estimated by the decrease of heat tolerance. Approximately, 40% of Bacillus coagulans and 70% of Bacillus licheniformis were germinated by HGCT for 120 min at 35 degrees C, respectively. Germination was confirmed by phase contrast microscopy. The effect of hydrostatic pressure treatment (HPT) at 6.5 MPa, 35 degrees C on the germination of B. coagulans and B. licheniformis spores were also investigated. Spores did not germinate by HPT alone at 6.5 MPa for 120 min.

Bacillus↗

PubMed1996

The leukocytes of the roughtail gecko Cyrtopodion scabrum: a bright-field and phase-contrast study.

The morphology of the peripheral blood leukocytes of the roughtail gecko, Cyrtopodion scrabum, is carefully described in Wright-Giemsa and toluidine-blue-stained blood films, and in the living condition by phase-contrast microscopy, using supravitally stained preparations. Mature eosinophils, basophils and small lymphocytes commonly occur in the blood, while monocytes are rarely seen. In addition, macrophages are occasionally encountered, but neutrophils cannot be observed. Developmental stages in eosinophil and basophil differentiation can be seen. This study serves as a basis for the cytochemical localization of substances within these blood cells.

Animals↗

PubMedDate not supplied

In vitro differentiation of rat seminiferous tubular segments from defined stages of the epithelial cycle morphologic and immunolocalization analysis.

Rat seminiferous tubule segments have been cultured in chemically defined medium (F12/DMEM 1:1) without added hormones or growth factors. The segments (1-2 mm) were isolated from defined stages of the cycle of the seminiferous epithelium (VIII and XII) by transillumination-assisted microdissection. The precise stages were examined by phase contrast microscopy of live cells squashed carefully out from the adjacent segments between glass slides. The squash technique was also used for a primary screening of the cultured tubules. Pachytene primary spermatocytes from stages VIII to XII of the cycle were able to complete meiotic divisions in vitro. From stage XII, they differentiated up to step 5 spermatids, expressed their specific antigens, and developed characteristic movement patterns of the flagellum and of the chromatoid body. Preleptotene and zygotene spermatocytes from the same cell association differentiated synchronously, as judged by chromosome morphology, characteristic chromosome rotation in zygotene and early pachytene, and by development of specific antigen expression. The elongation phase of spermiogenesis did not proceed normally in vitro. The rate of differentiation was the same as observed earlier in vivo. Earlier studies with [3H]thymidine labeling and autoradiography only permitted follow-up of the development of preleptotene spermatocytes. With the present method, all stages of spermatogenesis can be traced in culture with great accuracy in experiments relating to local regulation of spermatogenesis.

Animals↗

PubMed1994

Cytoprotection of human dermal fibroblasts against silver sulfadiazine using recombinant growth factors.

Topical antimicrobial agents, silver sulfadiazine (SSD) and mafenide acetate (MA), have been associated with delayed wound healing. Previous in vitro studies with human dermal fibroblasts (HDF) have shown progressive cellular cytotoxicity with increasing concentrations of SSD and MA. However, preexposure of HDF to epidermal growth factor, basic fibroblast growth factor, or platelet-derived growth factor has resulted in cytoprotection of HDF against 0.01 and 0.03% concentrations of SSD as determined by phase-contrast microscopy (PCM), hemocytometer cell counts, and total cellular protein content. PCM, however, showed slower destruction of HDF at the 0.05% concentration of SSD. These data suggest that cells activated by growth factors either take up less SSD or are more resistant to the direct cytotoxic effects of this drug.

Epidermal Growth Factor↗

PubMed1996

Carbohydrate- and CD18-dependent neutrophil adhesion to cardiac myocytes: effects of adenosine.

OBJECTIVE: Adenosine inhibits neutrophil adhesion and injury to isolated cardiac myocytes. In the present study, the contribution of selectin and CD18 interactions to neutrophil-myocyte adhesion and their sensitivity to adenosine were assessed. METHODS: Activated human neutrophils and canine myocytes were incubated with inhibitors of CD18 or selectin binding, adenosine, or combinations of both for 30-50 min at 37 degrees C. Neutrophils were pretreated with 0.1 microM fMLP for 10 min to study L-selectin-independent adhesion. Adhesion was measured by phase contrast microscopy. RESULTS: Anti-L-selectin mAb and the selectin-blocking carbohydrates sialyl Lewisx or mannose-6-phosphate, as well as anti-CD18 or anti-ICAM-1 mAbs, inhibited cell adhesion (by 84-99%, P < 0.05). CD11a, but not CD11b, was responsible for most of the CD18-mediated binding. An L-selectin-independent interaction between neutrophils and cardiac myocytes was observed that was delayed (peak adhesion at 40-50 min, rather than 30 min), but still inhibited by anti-CD18 mAb (by 65 +/- 11%, P < 0.05) and carbohydrates (by 87-97%, each P < 0.05). Adenosine (100 nM) inhibited this late CD18-dependent/L-selectin-independent phase of adhesion (by 61 +/- 14%, P < 0.05). The combination of adenosine and anti-CD18 mAb was additive such that adhesion was completely blocked (P < 0.05, compared to either agent alone). Inhibition of adhesion by adenosine was prevented by the A2 antagonist, DMPX (100 nM), and mimicked by the A2 agonist, CGS-21680 (10 nM) or the adenosine regulating agents, acadesine (100 microM) or GP531 (10 microM). CONCLUSION: Neutrophil-myocyte adhesion involved both L-selectin-dependent and L-selectin-independent carbohydrate binding as well as CD11a/CD18. Inhibition of adhesion by adenosine interferes with L-selectin-independent carbohydrate binding and possibly CD18.

Adenosine↗

PubMedDate not supplied

Validation of the morphologic end point of necrosis in rat hepatocytes subjected to oxyradical damage.

We have used phase-contrast microscopy to determine a necrotic end point of the order of minutes in primary hepatocytes exposed to oxyradicals generated with xanthine oxidase plus hypoxanthine. This study examines whether the morphologic end point thus determined agrees with other criteria of cell necrosis. When 95-100% of the cells were shown to be necrotic by our morphologic assay, transmission electron microscopy confirmed definitive subcellular evidence of cell death, trypan blue exclusion revealed a 92% loss in the ability of cells to exclude the dye, and there was a 47% specific release of 51Cr (versus a 50% theoretical value). In contrast, the appearance of extracellular aspartate aminotransferase activity was relatively slow and did not corroborate the morphologic end point. In summary, we have validated the morphologic end point in our cell-based assay of oxyradical damage.

Animals↗

PubMed1968

The circumfusion system for multipurposeculture chambers. II. The protracted maintenance of differentiation of fetal and newborn mouse liver in vitro.

The circumfusion system is a complex in vitro pumping unit incorporating 12 multipurpose culture chambers through which a serum-supplemented fluid nutrient is recirculated at a rate of 4.5 ml/min per chamber. This system was used to study the differentiative responses of fetal and newborn mouse liver explants placed in the serum-free environment formed between the sheets of unperforated cellophane and cover glasses of the chambers. Hepatocytes (parenchymal cells) were discernible in 3-5 days. They retained many of their features of differentiation in the circumfusion system for more than 120 days of cultivation. The living morphological characteristics of the hepatocytes were studied by phase-contrast microscopy (direct viewing and time-lapse cinemicrography) and by special cytochemical staining. Electron micrographs were made of both fresh liver specimens and the cultured cells. Comparisons of the cultured parenchymal cells with their in vivo progenitors showed a remarkable preservation of their differentiated state.

Animals↗