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PubMed1980

Evidence suggesting a role for cyclic nucleotides in acrosome reactions of hamster sperm in vitro.

There have been conflicting reports concerning the involvement of cyclic nucleotides in sperm capacitation. We have examined the effects of micromolar concentrations of dibutyryl cyclic AMP (Bt2cAMP) and of the phosphodiesterase inhibitors SQ20009 and ICI63,197 on hamster sperm incubated under in vitro capacitating conditions. Washed hamster sperm were incubated in a capacitation media containing bovine serum albumin, and a protein-free "motility-factor" from bovine adrenal cortex. Incubation for 3.5 hours was followed by addition of one of the compounds (0.1-10 microM) or control buffer. At the time of addition and after 30-120 minutes further incubation, sperm were examined by phase contrast microscopy. The final motility was similar to the initial motility (50-70%) and the same in incubation of controls or experimental compounds. Bt2cAMP, SQ20009, and ICI63,197 at these concentrations stimulated acrosome reactions to a statistically significant extent (P less than 0.005) compared to controls. Activation was stimulated to a varying degree by all three experimental compounds. These results suggest a role for cyclic nucleotides in capacitation and the acrosome reaction of hamster sperm.

Acrosome↗
PubMed1988

Formation of flagella during interphase in secondary spermatocytes from Xenopus laevis in vitro.

In cell culture, single motile flagella, 1 micron in length, were observed to grow from secondary spermatocytes of Xenopus laevis within 2-3 hours after telophase I, at 22 degrees C. About 90% of the secondary spermatocytes formed flagella as observed by phase-contrast microscopy. The flagella grew up to 2-6 microns in length during interphase II, which lasted about 18 hours. The presence of the "9 + 2" microtubular structure of the flagellar axonemes of secondary spermatocytes was confirmed by electron microscopy. When chromosomal condensation began (prophase II), the flagella were resorbed into the cells and, after the second meiotic division, a flagellum was formed again by each of the round spermatids. Thus, there appears to be a close relationship between the meiotic division cycle and the formation of flagella. The possible contribution of Sertoli cells to the formation of flagella in secondary spermatocytes was examined by reducing the number of Sertoli cells to less than ten per culture. Under these conditions, flagella formed in secondary spermatocytes with very high efficiency. It is very likely that secondary spermatocytes form flagella in vivo, since the secondary spermatocytes were observed to have flagella immediately after dissociation of the testes.

Animals↗
PubMed1994

Cytokines modulate terminal differentiation and the expression of predesquamin in cultured keratinocytes.

Predesquamin is a glycoprotein that we have isolated from human epidermis. Its monospecific polyclonal antibody immunolocalizes to the lower stratum corneum. We have previously found that it is not expressed in primary keratinocyte cultures unless both interferon-gamma and serum are added to the culture media. We now show similar results with interleukin-1 alpha substituted for interferon-gamma (but not with retinoic acid). Predesquamin is expressed in media supplemented with both cytokines (with or without serum), accompanied by the observation of larger outer squames and a more mature pattern of desquamation. As both cytokines are naturally present in the epidermis, this suggests that they contribute to desquamation in vivo. The predesquamin expression is demonstrated by Western blots of cell extracts with the specific antibody. Corresponding morphological changes are illustrated by phase-contrast microscopy.

Blotting, Western↗
PubMed2000

Adhesion of lymphoid cells to CD44-specific substrata: the consequences of attachment depend on the ligand.

Interactions between cell-surface adhesion receptors and immobilized specific substrata can exert profound effects on cell morphology. Using phase-contrast microscopy, we show that CD44-expressing mouse lymphoid cells display a spread morphology when adhering to CD44-specific monoclonal antibody (mAb) immobilized on plastic. This spread morphology is different from that of these same cells when adhering to immobilized hyaluronan, the natural ligand of CD44. Morphometric measurements, in combination with intracellular actin staining and fluorescence microscopy, revealed that the adhesion of lymphoid cells to hyaluronan required essentially no cytoskeletal reorganization and resulted in no fundamental change in morphology. On the other hand, cells adhering to immobilized CD44-specific mAb rearranged their actin structure and established multiple membrane contact sites (spread). Cell spreading on antibody, but not attachment to hyaluronan, was inhibited by cytoskeleton-disrupting agents. Transfection of CD44-negative lymphoid cells with full-length and tailless CD44 enabled these cells to bind to both immobilized hyaluronan and mAb. However, the transfectant lacking the cytoplasmic tail of CD44 spread only transiently on the antibody-coated surface. Our results suggest that CD44 may mediate lymphocyte attachment to its carbohydrate ligand hyaluronan by mechanisms broadly similar to those used by selectins. When immobilized CD44-specific antibody is the ligand, however, CD44 may regulate the activity of the cytoskeleton by mechanisms broadly similar to those used by integrins. In the latter case, the cytoplasmic domain of CD44 contributes to cell spreading.

Actins↗
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↗
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↗
PubMed2002

Use of vascular explants for ex vivo neovascularization of biomaterials.

Biomaterial polymers have been proposed as scaffolds for cell assembly in vascular bioengineering. We describe here a new method for the neovascularization of polyurethane meshes from explants of rat aorta. Aortic rings embedded in collagen-permeated polyurethane meshes and cultured in medium supplemented with fetal bovine serum and vascular endothelial growth factor generated florid microvascular outgrowths that efficiently vascularized the available spaces between polyurethane fibers. The neovessels could be identified in the live cultures by phase-contrast microscopy, and in formalin-fixed preparations by the ABC peroxidase procedure, using the endothelial-specific Griffonia isolectin B4. The aortic outgrowths were successfully labeled with the intravital fluorescent dyes Calcein AM or SPDiOC(18), which are nontoxic and can be used for tracking studies. This study shows that artificial biomaterial meshes can be colonized ex vivo with histotypic microvascular networks, and provides the proof of concept for the future development of stably vascularized devices for in vivo implantation.

Animals↗
PubMed1995

Anabolic-androgenic steroid-induced toxicity in primary neonatal rat myocardial cell cultures.

Recent literature reports of myocardial infarction in athletes who self-administer anabolic-androgenic steroid (AAS) and previous animal studies of the effects of AASs on the heart suggest that these drugs may be directly injurious to the myocardium. We have previously demonstrated that 100 microM testosterone cypionate (TC) inhibits all beating activity of primary neonatal rat myocardial cell cultures within 1 hr of exposure and causes significant LDH release by 4 hr of exposure, indicating a direct toxic effect of TC. The purpose of this investigation was to evaluate the effects of commonly abused AASs on primary neonatal rat myocardial cell cultures and to provide insight into early cellular changes that may lead to TC-induced toxicity. Significant LDH release was observed in 5-day-old primary myocardial cell cultures (obtained from 3-to-5-day-old Sprague-Dawley rats) exposed to 100 microM testosterone enanthate (TE), testosterone propionate (TP), and oxymetholone (O) for 4 and 24 hr and in cultures exposed to 100 microM testosterone (T) for 24 hr. Neutral red retention and MTT formazan production were significantly decreased in cell cultures exposed to 100 microM TE, TP, and O after only 4 hr of exposure, indicating a loss of viability and mitochondrial activity. However, there was no effect on viability of cell cultures exposed for 24 hr to 100 microM of a variety of other commonly abused AASs. Phase-contrast microscopy revealed complete disruption of the monolayer in cell cultures treated with 100 microM TE, TP, and O for 4 hr. Treatment of fura-2-loaded myocardial cell cultures with 100 microM TC produced no significant changes in calcium transients or baseline calcium levels for up to 13 min of exposure. These results indicate that O, T, TC, TE, and TP produce a direct toxic effect in heart cell cultures and that early (< 13 min) changes in calcium homeostasis are unlikely to participate in the mechanism of toxicity.

Anabolic Agents↗
PubMed1991

In vitro viability of choroidal melanomata following pre-enucleation irradiation.

In a group of eighteen patients with uveal melanomas, seven underwent low-dose pre-enucleation irradiation of approximately 2000 cGy. All the tumours were propagated in tissue culture and the growth characteristics of tumour cells from irradiated eyes were compared with tumour cells from non-irradiated eyes. Cultures were observed with phase-contrast microscopy, and radioactive thymidine labelling was used to study cell turnover. Although tissue samples from peripheral areas of irradiated tumours produced a mixture of viable and non-viable cells, with reduced ability to attach to substrate, central regions of irradiated tumours contained viable cells which propagated freely in tissue culture.

Cell Differentiation↗
PubMed1995

A threshold decrease for electrically stimulated motor responses of isolated aging outer hair cells from the pigmented guinea pig.

When outer hair cells are isolated from guinea pig cochleas and are placed in normal Hank's medium, they exhibit aging as a slow tonic reduction in length and increase in diameter. During this time the lateral subsurface cisternae become progressively vesiculated and the optical density of the border seen under phase-contrast microscopy decreases. A study of 65 outer hair cells was carried out using video imaging of this process. The base of each cell bonded to the Petri dish and the motility of the cuticular plate was recorded in two ways. To quantify the slow contraction of each preparation, the dimensions of the cell were measured from video replay. Displacements of the cuticular plate in response to an alternating electric field in line with the cell axis were also monitored using a video tracking technique. The amplitude of a 1 Hz stimulus required to cause a visually detectable motor response above baseline noise decreased as the cell degraded. Typically, fresh cylindrical cells exhibiting high optical contrast showed relatively small movements for field strengths up to 2 kVm-1. However, as the cell depolarized, the rigidity initially decreased and the cell could respond to field strengths down to 0.1 kVm-1 before cell death ultimately occurred. Such a threshold phenomenon in the isolated OHC has not been demonstrated directly until now. This result explains the variability of electromotility in aging in vitro preparations from the cochlea.

Animals↗
PubMed1994

Cytomorphological, cytogenetic, and molecular biological characterization of four new human renal carcinoma cell lines of the clear cell type.

Four new permanent cell lines (RCC-A, -B, -C, and -D) derived from different human renal cell carcinomas of the clear cell type were established in tissue culture. The cell lines displayed characteristic differences in cell size and shape, which allowed individual identification by phase contrast microscopy. Ultrastructurally, the cell lines exhibited varying amounts of cytoplasmatic glycogen and lipid. Immunohistochemistry revealed co-expression of vimentin and cytokeratin in all cell lines. The mean population doubling time ranged from 27 h (RCC-A) to 104 h (RCC-D). RCC-B and -C cells produced slowly growing tumours after heterotransplantation into nude mice, whereas RCC-A and RCC-D cells were non-tumorigenic. The modal chromosome number was either near-diploid (RCC-A, -B, and -C) or near triploid (RCC-D). Clonal abnormalities affecting the short arm of chromosome 3 were seen in all cell lines. Northern blot analysis revealed no expression of the proto-oncogenes c-fos, c-ros, and c-mos, whereas c-Ki-ras expression was observed in all cell lines. Expression of c-myc was observed in RCC-A, RCC-B, and RCC-D cells, whereas c-raf expression could be detected in RCC-B and RCC-D. Tumour suppressor gene p53 mRNA was observed in the cell line RCC-D.

Adenocarcinoma, Clear Cell↗
PubMed1994

Ultrastructural localization of myoglobin mRNA in human skeletal muscle.

The intracellular localization of myoglobin mRNA in the skeletal muscles of normal subjects was examined by in situ hybridization using a biotin-labeled cDNA probe. By phase-contrast microscopy, myoglobin mRNA signals were demonstrated to be located preferentially on the A-band. Two different methods of tissue preparation, i.e., pre-embedding method and post-embedding method, were used for the electron microscopic study. With the pre-embedding method, only a few gold particles were found to be associated with cytoskeletal filaments in the intermyofibrillar space. With the post-embedding method, superior preservation of sections and higher signal intensities were obtained. Although most of the gold particles were localized on the A-band, some were seen in other regions; i.e., in the intermyofibrillar space, perinuclear space, or the I-band, where myoglobin is localized. These findings suggest that myoglobin is primarily synthesized on the A-band, where ribosomes predominantly exist, although myoglobin is also localized on the I-band. The predominant localization of myoglobin mRNA on the A-band may aid in the mRNA transcription and may be related to the regulation of myoglobin synthesis in skeletal muscle cells.

Adult↗
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↗
PubMed1975

Antibiotic-resistant mutants of Bacillus subtilis conditional for sporulation.

Among spontaneously occurring antibiotic-resistant mutants of Bacillus subtilis 168 we have identified a sub-class that is conditionally sporulative. Mutants in this sub-class are resistant to antibiotic during vegetative growth but are sensitive during sporulation. Mutants conditionally-resistant to erythromycin, kanamycin, spectinomycin, and streptomycin have been isolated and characterized by phase contrast microscopy and with respect to their ability to synthesize heat-resistant endospores or the sporulation-associated enzyme alkaline phosphatase. The results suggest that several entirely different genetic lesions may result in this single phenotype. This group includes mutants whose properties suggest that both th 30S and 50S ribosomal subunits may be altered concomitant with early spore specific metabolism. The blockage imposed by antibiotic may be at or near Stage 2 of sporulation.

Anti-Bacterial Agents↗
PubMed1990

Lactate dehydrogenase release as an indicator of dithranol-induced membrane injury in cultured human keratinocytes. A time profile study.

HaCaT cells, a rapidly multiplying human keratinocyte line, were tested for their sensitivity to antipsoriatic dithranol with regard to classical proliferation parameters and for the drug's action on the plasma membrane integrity by the dose- and time-dependent release of cytosolic lactate dehydrogenase (LDH). In the case of 3H thymidine as well as 14C amino acid incorporation the 50% inhibition concentration (IC50) was 0.2 microM dithranol 24 h after initial exposure to the drug. For protein content of attached cells the IC50 proved to be greater than 3.0 microM. Using 0.3, 1.0 and 3.0 microM dithranol, significant (p less than 0.05) dose dependent LDH release of 0.866 +/- 0.387, 1.842 +/- 1.127 and 2.938 +/- 1.635 mU per hour and cm2 confluent culture area was measured between the 5th and the 24th hour, compared to an acetone control of 0.504 +/- 0.299 mU/h x cm2. Between the 2nd and the 4th hour as well as from the 25th to the 48th hour and the 49th to the 72nd hour the LDH release after dithranol treatment did not exceed the control value. In accordance with these findings dose-dependent morphological signs of cell injury were detected by phase contrast microscopy beyond the 4th hour. The data reveal that: HaCaT cells are a very sensitive target for the antiproliferative action of dithranol; the drug causes considerable plasma membrane damage even at concentrations as low as 0.3 microM; and this membrane damage becomes evident after a latency of at least 4 h and for a limited period of up to 24 h.

Anthralin↗
PubMed1980

Direct studies of dikaryotization in Schizophyllum commune. I. Live inter-cellular nuclear migration patterns.

Compatible matings of Schizophyllum commune were performed on glucose-peptone-yeast extract medium appended with gelatin (18%) and studied by phase contrast microscopy during nuclear migration. Three categories of nuclear migration were observed. Type I involved a pulsatile jerking of the entire cytoplasmic contents of the hypha, changed direction periodically, and, during periods of cytoplasmic tranquility, the nucleus continued to migrate. Type II A migration of nuclei occurred in the absence of visible cytoplasmic flow. Both Type I and Type II A nuclear movements exceeded the hyphal growth rate by 10--20-fold. Type II B nuclear migration also occurred in the absence of visible cytoplasmic flow and the velocity was within the range of the hyphal growth rate. No specific organelles that were detected either directed or facilitated Type II A or Type II B nuclear movements. The nucleolus could either lead or trail relative to the direction of nuclear movement. Nuclear migration can be attributed to both cytoplasmic flow and self motility, depending upon the particular regions of the migration hypha in which it occurs.

Agaricales↗
PubMed1986

A method for standardizing cytologic sampling for the estimation of nasal ciliary activity.

A method for sampling cytological brushings of human nasal mucosa using phase-contrast microscopy is detailed and the modes for counting the ciliary beat frequency (CBF) are reviewed. To test the reproducibility of our method, we performed two sampling procedures on subsequent days on each of 19 volunteers and patients. The total volume of harvested cells, the number of beating ciliated cells, and the CBF were assessed. Since there are some differences present in subsequent cell samples, our findings indicate that it may be better to critically evaluate a single sampling procedure in order to establish a correct clinical diagnosis, especially if the number of harvested cells is small. The need to standardize methods for counting the CBF to achieve comparable results is stressed.

Cilia↗
PubMed1979

Effects of lithium on morphological characteristics of dissociated brain cells in culture.

Lithium chloride was added in 5 and 10 mM concentrations for different periods of exposition time to dissociated cultures obtained from chicken embryonic brain. When supplementing lithium at day 1 in vitro for five days, a dose-dependent decrease in total protein was observed in the cultures as compared to the sodium-treated controls. Profound reduction was revealed in the length of neuronal processes and in the number of neuronal cell bodies by phase contrast microscopy and by morphometric means. After exposition of lithium in 10 mM concentration for 48 h, beside a slight decrease in number of perikaryons, a marked reduction in process length of neural elements was observed in the 6-day-old tissue cultures. Ultrastructurally, swollen and degenerating nerve processes have been found after lithium treatment suggesting a particular sensitivity of these structures to lithium ion.

Animals↗