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PubMed1994

I-131-lipiodol cytotoxicity in hepatoma cells.

Hepatoma is a common cancer in Taiwan. New effective treatment for hepatoma patients is urgently needed. Encouraging results of I-131-lipiodol treatment for hepatoma with minimal toxicity have been recently reported. The mechanism of lipiodol targeting and retention by hepatoma are not well understood. The cellular interaction of lipiodol and the cytotoxic effects of I-131-lipiodol on hepatoma cells were investigated in this study. HepG2 cells were cultured with lipiodol, and untreated HepG2 cells were used as the control. Changes of cellular morphology were accessed by light and electron microscopy. The uptake and retention of lipiodol by HepG2 cells were studied by phase contrast microscopy and neutron activation analysis. HepG2 cells were cultured with I-131-lipiodol varying from 0.12 microCi to 120 microCi. The cytotoxic effect of I-131-lipiodol was evaluated by the surviving fraction of HepG2 cells. Changes in cellular morphology was examined by light microscopy. Results indicated that HepG2 cells were capable of active uptake of large amounts of lipiodol by endocytosis and prolonged intra-cellular retention associated with the formation of many bulging cytoplasmic extensions. I-131-lipiodol was highly cytotoxic to HepG2 cells. There was a steep dose response relationship, and the effective dose (LD50) was 1.2 microCi (480 rads). The cytotoxic effects of I-131-lipiodol were associated with pleomorphism of HepG2 cells, an increase in cell size and nuclear-cytoplasmic ratio, an increase in the size and number of nuclei, and vacuolation of the cytoplasm around the nuclear regions. Multiple nucleoli, fragmentation and segregation and ring shaped changes of nucleoli were also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Hepatocellular↗
PubMed1981

Identification of living spermatogenic cells of the mouse by transillumination-phase contrast microscopic technique for 'in situ' analyses of DNA polymerase activities.

The stages of spermatogenesis can be identified in freshly isolated, unstained adult mouse seminiferous tubules using a transillumination method. Late acrosome- and maturation phase spermatids, arranged in bundles at stages XII-VI give rise to a spotty transillumination pattern. Before spermiation, these cells form a continuous layer on the top of the seminiferous epithelium, recognized by a strong homogeneous central light absorption in the freshly isolated seminiferous tubules at stages VII and VIII. Other stages have a pale light absorption pattern. The accurate determination of the developmental stages of the germ cells was based on the morphology of the developing acrosomic system and of the nuclei of the spermatids, as revealed by phase contrast microscopy. Using this procedure, the activity levels of DNA polymerases alpha and beta have been studied by autoradiography of squash preparations. Using endogenous templates, assay conditions that differentiate between the solubilized DNA polymerases alpha and beta in vitro, were used to distinguish between these activities in situ in different stages of mouse spermatogenesis. Except in very late spermatids shortly before spermiation, DNA polymerases alpha and beta were detectable in all cell types examined. Coinciding with the nuclear protein transitions, elongating spermatids at steps 10-12 and maturation phase spermatids at steps 13-14 showed high DNA polymerase activities. As no replication occurs in these cells, the observations support the view that both DNA polymerases alpha and beta could be involved in repair DNA synthesis.

Animals↗
PubMed1991

Morphological and functional similarities between cultured prostatic stromal cells and testicular peritubular myoid cells.

A number of androgen effects on epithelial cells may be mediated by androgen-regulated paracrine factors produced by underlying mesenchymal cells. In previous studies we demonstrated that prostatic stromal cells and testicular peritubular cells, derived from immature rats, produce mediators of androgen action with identical effects on Sertoli cells. In the present paper we further compared the morphological and functional characteristics of both mesenchymal cell types. Cultured prostatic stromal cells and testicular peritubular cells look identical under phase-contrast microscopy, share the ability to form tubular structures and "balls" when cocultured with Sertoli cells, and contain proteins immunoreactive with an antiserum against alpha-smooth muscle isoactin. Two-dimensional gel electrophoresis shows that the pattern of proteins produced by both cell types is nearly identical. Conditioned media from stromal and peritubular cells contain a factor that stimulates transferrin and cGMP production in Sertoli cells. The behavior of the active principle in the media from both cell types is comparable. On reverse-phase HPLC the elution profile of this factor is comparable for media from both cell types. In conclusion, these data point to a striking similarity in the morphological and functional characteristics of mesenchymal cells cultured from the prostate and testis.

Animals↗
PubMed1983

A rationale for the management of periodontal diseases: effects of tetracycline on subgingival bacteria.

Microbiologic criteria obtained with phase-contrast microscopy were used in a short-term, double-blind study to measure the effects of systemic tetracycline HCl on subgingival bacterial populations in advanced periodontal pockets refractory to local therapy (repeated scaling, root planing, and the subgingival administration of chemotherapeutic agents [H2O2, NaHCO3, NaCl, MgSO4]). Twenty-one subjects, selected for study, had at least one of the following conditions present after local therapy: spirochetes, motile rods, or crevicular leukocytes greater than or equal to 125 per phase-contrast microscopic field. Tetracycline HCl (1 gm/day for 14 days) was randomly distributed to 11 subjects and a placebo to ten subjects, so that neither the subjects nor investigators were aware of the prescription contents. Evaluations after two weeks disclosed that tetracycline HCl significantly reduced elevated levels of spirochetes, motile rods, and crevicular leukocytes to low or undetectable levels, whereas levels in the placebo subjects remained generally unchanged. The results clearly demonstrate the value of tetracycline HCl as an adjunct to periodontal therapy in reducing remaining suspected periodontopathic bacterial populations in advanced lesions after local therapy of scaling, root planing, and topically applied chemotherapy.

Adult↗
PubMed1994

Cytotoxic effects of FK506 on human renal proximal tubule cells in culture.

FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200 micrograms/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40 micrograms/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.

Cell Survival↗
PubMed2006

Pore size characterization of monolith for electrochromatography via atomic force microscopy studies in air and liquid phase.

This paper investigates the use of scanning electron microscopy (SEM) and atomic force microscopy (AFM) for the characterization of monoliths used in capillary electrochromatography (CEC) while focusing on the nature of the information available from both techniques. SEM imaging revealed a compact structure of non-porous micrometer sized particles homogeneously agglomerated. With a simple AFM methodology, we found by direct observation that the same material exhibits mesopores in the nanometer range while SEM showed non-porous surfaces. These results obtained by AFM clearly showed that micrometer sized particles shrank and micrometer sized pores increased in the monolith when wetted. Thus, AFM was capable of demonstrating the morphological differences between wet and dried monolithic materials that are not possible by other imaging methods at micrometer resolution.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2001

Conventional optical microscopy of colloidal suspensions.

Optical microscopy can resolve detail at the larger end of the colloidal length scale, and to image suspensions at an individual particle level of resolution would allow the investigation of local behaviour in a way denied to the established scattering techniques. However, to achieve high-contrast single-particle resolution in dense suspensions that are thick enough to show behaviour the same as would be expected in the bulk is not a trivial exercise. We build on established advanced techniques of the conventional (i.e. non-confocal) light microscopy of phase objects to develop a suitable experimental protocol. Furthermore, we demonstrate the effectiveness of this protocol by means of an 'atlas' of the hard-sphere crystalline solid (where random stacking results in many complex facets), which should serve as a compendium for future study.

Bone Cements↗
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↗
PubMed1993

Simultaneous and separated culture of keratinocytes and fibroblasts on each side of a collagen membrane.

We have developed a new cell culture system which is of benefit for the research of dermal-epidermal interaction. In this system, normal human keratinocytes are cultured on the upper surface of a permeable collagen membrane, on the undersurface of which human fibroblasts are simultaneously and separately cultured in a lifted condition. Both the keratinocytes and fibroblasts showed good proliferation and differentiation which were revealed by phase contrast microscopy as well as light and electron microscopies. A permeability test of the collagen membrane used in the present study showed that peptides of less than 30 kDa are able to penetrate through the membrane. Using this system, we estimated the total protein content and cornified envelope formation in the keratinocytes cultured with or without fibroblasts. We found that the total protein and cornified envelope formation were significantly increased when keratinocytes were cultured together with fibroblasts. When keratinocytes were cultured with fibroblasts in the condition of air-liquid interface, synthesis of the cornified envelope was further enhanced. These results indicate that fibroblasts really effect not only the proliferation but also the differentiation of keratinocytes, and the air-liquid interface enhances their effect on differentiation. This bi-phase separated cell culture system is a useful tool for the study of keratinocyte-fibroblast interaction.

Cells, Cultured↗
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↗
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

Clonal analysis of expression of epithelial antigens in cultures of normal human breast.

Cells from normal human breast epithelium were cloned in monolayer culture and the clones were stained with monoclonal antibodies. Tissue was from reduction mammoplasty operations. Cloning efficiencies were 5-30%. Two types of clone were identified: 10 to 30% were of relatively spread cells whose boundaries were often difficult to see by phase-contrast microscopy but where they were visible they appeared as dark lines. The edges of the clones usually appeared to be under tension. These clones were stained by two monoclonal antibodies, LICR-LON-M8 and M24, that stain luminal epithelial cells in the intact tissue, but not myoepithelial or stromal cells. Within a clone the cells showed a full range of antigenic phenotypes. This was confirmed for clones grown from single cells that had been isolated manually. The second type of clone was more compact with little evidence of tension at the edges, and cell boundaries were clearly visible and bright under phase contrast. These clones were not stained by antibodies M8 or M24. Both types of clone stained with a third monoclonal antibody that is specific for luminal epithelial cells in the intact tissue, LICR-LON-M18, but the distribution of staining was different in the different types of clone. The simplest interpretation of the two types of clone is that luminal epithelial cells give rise to the spread type of clone while the myoepithelial cells give rise to the more abundant and vigorous compact clones. Alternatively, the compact clones may be from luminal epithelial cells that have lost differentiated characteristics.

Antibodies, Monoclonal↗
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↗
PubMed1997

A coherent phase diagram of the [(AuCu)0.86Ag0.14]1-XPtX pseudobinary section in Au-Cu-Ag-Pt quaternary system.

In order to determine a coherent phase diagram of the [(AuCu)0.86Ag0.14]1-XPtX pseudobinary system, phase identifications and analyses of the microstructural configurations were performed by means of transmission electron microscopy. Various phase regions were detected in the coherent phase diagram as follows; (1) alpha 0 (fcc) single phase region, (2) two-phase region (alpha 1 + alpha 2), (3) AuCu I (L10) + alpha 0, (4) AuCu I + alpha 2, (5) AuCu II (L10-S) + alpha 2, (6) AuCu I + AuCu II + alpha 2, (7) AuCu I single phase region. Microstructural features of these regions relating to the phase transformations were described.

Chemical Phenomena↗
PubMed1986

Movements and other distinguishing features of small vesicles identified by darkfield microscopy in living macrophages.

Perinuclear vesicles (estimated diameter less than 0.15 micron), too small to be seen in living mouse macrophages by direct phase-contrast microscopy, could be detected by darkfield microscopy thanks to their rapid non-saltatory movements at 37 degrees C, contrasting with the slower saltations of accompanying phase-visible larger vesicles (0.25-0.5 micron, presumed secondary lysosomes). The movements of these 'small visicles' also differed from those of the 'larger visicles' in their responses to changes in temperature, and to chemical agents known to inhibit both the saltations of secondary lysosomes and the latter's fusion with phagosomes. Thus the 'larger vesicles' stopped moving at 25 degrees C, the small ones did not; both stopped at 18 degrees C. The 'small vesicles' continued to move actively after cell uptake of the polyanion poly-D-glutamic acid, while the saltations of the 'larger vesicles' were markedly slowed; both sets of vesicles stopped after uptake of ammonium chloride. Degranulation of the small vesicles paralleled that of the larger, while simultaneously observed preformed pinosomes (labelled with fluorescent wheat germ agglutinin (WGA) appeared to be unaffected. On the basis also of refractivity, location and speed the 'small vesicles' are considered not to be pinosomes, but probably to be lysosomes. The question of whether they are a subgroup of small immature secondary lysosomes or primary lysosomes (0.05-0.08 micron) is discussed. The broad spectrum of movement inhibited by ammonia in macrophages raises the possibility that this weak base inhibits movements of all lysosomes. Further characterization of these 'small vesicles' requires their relation to be defined to the small particles in other cell types (especially in axoplasm) which have been detected by video-enhanced microscopy.

Ammonium Chloride↗
PubMed2001

Protein kinase C alpha and gamma in N/N 1003A rabbit lens epithelial cell differentiation.

PURPOSE: To determine if Protein Kinase C (PKC) plays a role in the initiation of lens epithelial cell differentiation into a lens fiber cell. METHODS: PKCalpha or PKCgamma was overexpressed in N/N 1003A lens epithelial cells for up to 7 days. Phase contrast microscopy was used to observe morphological changes associated with PKCalpha or PKCgamma overexpression. Cell cycle changes in cells overexpressing PKCalpha or PKCgamma were measured using acridine orange staining and flow cytometry. Crystallin levels in cells overexpressing PKCalpha or PKCgamma were measured using Western blots and RT-PCR. RESULTS: Significant differences in cell cycling were observed between untransfected cells and those overexpressing PKCalpha or PKCgamma. Overexpression of PKCalpha and PKCgamma caused the cells to lose their epithelial-like appearance and elongate. alphaB-crystallin expression was detected in all the samples while alphaA-crystallin was detected only in cells after 7 days of PKCalpha or PKCgamma overexpression. CONCLUSIONS: The observations that alphaA-crystallin is only found in N/N 1003A cells overexpressing PKCalpha or PKCgamma for 7 days along with the finding that a block in the G0/G1 phase of the cell cycle and the consequent morphological changes are observed, indicate that PKCalpha and PKCgamma may have a role in the initiation of differentiation in lens epithelial cells.

Acridine Orange↗
PubMed1991

Distribution, morphology and epithelial interactions of bovine spermatozoa in the oviduct before and after ovulation: a scanning electron microscope study.

In cows undergoing spontaneous oestrous cycles and mated during the first 6 hours of oestrus, the distribution of spermatozoa in the oviduct isthmus and changes in their surface membranes and neighbouring epithelium have been examined shortly before and after ovulation. In agreement with previous histological studies, relatively few spermatozoa were detected in the oviduct lumen: most were located in the caudal isthmus before ovulation, frequently among folds and in the presence of a viscous secretion. A majority of spermatozoa in this region showed strands and droplets of secretory material distributed over the anterior portion of an intact head before ovulation, whereas distribution of material over the post-nuclear cap of spermatozoa close to vesiculation or already acrosome-reacted was characteristic of the post-ovulatory situation. These changes in sperm head membranes were viewed as an expression of the completion of capacitation, and seemingly permit microvillous engagement with the rostral tip of the head. In conjunction with a narrow lumen and viscous secretions in the caudal isthmus, microvilli may thus serve to regulate periovulatory sperm progression towards the site of fertilisation, and be the basis of intermittent phases of adhesion to the oviduct epithelium as seen by phase-contrast microscopy. Although cilia do not similarly engage the heads of bull spermatozoa (cf. boar spermatozoa), they may act to regulate progression of capacitated spermatozoa by contacting the principal piece of the flagellum. In the light of these observations, changes in the molecular composition of sperm surface domains during the process of capacitation in vivo now require specific definition.

Animals↗