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PubMed1993

Establishment and characterization of a rat yolk sac tumor cell line, NMT-1, producing alpha-fetoprotein, with potential for lymphatic metastasis.

A new cell line producing alpha-fetoprotein, designated NMT-1, was established from rat yolk sac tumor with the potential for lymphatic metastasis in inbred Wistar rats. In this paper, we investigated the characteristics of NMT-1 cells. The cell line grew in a monolayer and had a polygonal epithelioid appearance in phase-contrast microscopy. The doubling time of NMT-1 cells during exponential growth was approximately 16 h in RPMI-1640 with 10% fetal calf serum. The D0 value for radiation sensitivity was 97 +/- 3 cGy. The extrapolation number, n, for NMT-1 was 1.08 +/- 0.15. In the log phase, the G0/G1, S and G2/M fractions were 23.2%, 62.1% and 14.3%, respectively. Tumor take was observed in all of the rats inoculated with NMT-1 cells. In the case of flank tumor, the mean survival time was 104 days after inoculation with 10(6) tumor cells. Inguinal, axillary, paraaortic, mesenteric and mediastinal lymphnode metastases were observed in all rats inoculated with NMT-1 cells. Rats which survived for a long time developed metastases in a lymphatic vessels of the liver, lung and/or kidney microscopically. The biological behavior and the histopathological features of the tumor induced by inoculation with NMT-1 cells were the same as those of the original tumor induced by fetectomy.

Animals↗
PubMed1967

Differential expression of bacteriophage genomes in vegetative and sporulating cells of Bacillus subtilis.

Two antigenically distinct bacteriophages, beta3 and beta22, have been isolated and characterized with Bacillus subtilis strain W23 as a host. They differ in plaque morphology, single-step growth characteristics, host range, and thermal stability. The deoxyribonucleic acids isolated from beta3 and beta22 differ in base composition, density in CsCl and Cs(2)SO(4), sedimentation coefficient, molecular weight, and thermal denaturation temperature. These phages have been used to analyze the ability of B. subtilis to sporulate despite infection by virulent phages. When development of phages beta3 and beta22 in sporulating cultures was compared with that in log cultures, an increase in the latent periods of infection and a decrease in the burst sizes for the two phages were observed. Sporulating cultures infected with beta3 yielded the usual percentage (85%) of mature spores; 80% of these contained phage determinants and 20% were uninfected. However, cultures infected with beta22 lysed. Of the small fraction (0.01%) which sporulated, 83% were uninfected and 17% were infected. Phage beta3-infected and uninfected spores were examined to distinguish any chemical or physical differences. Preparations of both types of spore contained 81.4 mug of dipicolinic acid per mg (dry weight), and examination by phase-contrast microscopy gave no evidence of any difference in outward appearance. A 20% decrease in infected spore count was observed upon heating at 80 C for 10 min. Differences in the infection processes of the two phages prompted an analysis of the transcription process after infection. Deoxyribonucleic acid-ribonucleic acid hybrid analysis of relative amounts of phage-specific and host-specific messenger ribonucleic acid (mRNA) present in infected cells suggested that beta3 was unable to repress the synthesis of host mRNA and that beta3-specific mRNA synthesis was repressed in sporulation-phase cultures. Phage beta22, in contrast, was able to repress host-specific mRNA synthesis in both log-infected and sporulation-infected cells. The results suggest that the differential expression of the phage genomes is due to the relative ability of the phages to repress the host genome.

Bacillus subtilis↗
PubMed1975

[The application of interference contrast microscopy in biology (author's transl)].

Like phase contrast, Nomarski interference contrast microscopy can be used to examine unstained specimens in biology and medicine. The properties of both contrast enhancement techniques are illustrated by various examples. The phase contrast method is especially suited for thin specimens with small differences in refractive index, whereas the interference contrast method supplies good results even of thick specimens. Interference contrast is a valuable supplement to the phase contrast method, and expands the application of microscopy in biology

Animals↗
PubMed2004

The effect of periodontal treatment on the salivary bacterial load and early plaque formation.

AIM: The purpose of the present study was to investigate in periodontitis patients the relationship between the number of bacteria in the saliva and the amount of de novo plaque formation before and after treatment. METHODS: At baseline, before any treatment was provided, patients rinsed with 10 ml sterile saline. After professional tooth cleaning the patients were instructed to abolish all tooth cleaning procedures for the subsequent 24 h. After this period, the rinsing procedure was repeated and the amount of de novo plaque was assessed. Three months after the initial periodontal therapy was completed the experiment was repeated. The microbiological evaluation of the rinsing samples was carried out by means of phase contrast microscopy and anaerobic culturing. RESULTS: After treatment the amount of de novo plaque was less compared to before treatment, 0.40 and 0.65 respectively. Both before and after treatment more de novo plaque was present at sites with inflammation than at healthy sites. In order to evaluate the contribution of the numbers of salivary bacteria to the amount of de novo plaque formation an analysis was carried out for healthy sites. This analysis included only healthy sites as determined before treatment and the same sites after treatment. The results showed a significant reduction in the de novo plaque formation after treatment (0.49 before and 0.22 after treatment). Phase contrast microscopic evaluation showed that the number of bacteria in the rinsing samples after treatment was less than before treatment. After treatment also a reduction was found in the prevalence of Prevotella intermedia, Tannerella forsythensis and Peptostreptococcus micros in the rinsing samples. CONCLUSION: The present study confirms the observation in the literature that the periodontal condition is of major importance in the rate of de novo plaque formation. In addition, the results suggest that the number of bacteria in the saliva may play a role.

Adult↗
PubMed1999

Study of single-molecule dynamics and reactions with classic light microscopy.

Single-molecule studies in the life sciences often deal with observation or spectroscopy. Studies of reactions are rare, and the light microscope has been used for such experiments only occasionally. In an experimental environment, for example, as is required for most nearfield scanning or electron microscopies, it is difficult to study single-molecule reactions of biological relevance. Therefore, we have developed techniques to study single-molecule reactions with classic (nonscanning) farfield light microscopy. The conversion of nicotinamide adenine dinucleotide (NAD+) and lactate to NADH (a reduced form of NAD+), pyruvate, and H+ catalyzed by a few LDH-1 enzyme molecules has been studied in substrate solutions with different viscosity using the NADH autofluorescence. It is even possible to monitor the progress of the reaction by phase-contrast microscopy via scattering or absorption by product molecules. As an example for a single-molecule reaction with a macromolecule as substrate, the handling and enzymatic cutting of fluorescently stained lambda-DNA is studied. In solutions containing 10 mM magnesium and 66 mM potassium ions at pH 7.9, an individual DNA molecule tends to collapse into a globular structure. When moved through an aqueous solution, it becomes stretched by viscosity drag. After stopping the motion, the molecule collapses and the dynamics of this process can be quantified. When a restriction enzyme is present, sequence-specific cutting can be directly observed in the light microscope. The theoretical restriction pattern, as predicted from the sequence of the molecule, can be generated directly under visual inspection.

Bacteriophage lambda↗
PubMedDate not supplied

Flow cytometric DNA analysis of defined stages of rat seminiferous epithelial cycle during in vitro differentiation.

The stages of the rat seminiferous epithelial cycle have been isolated for flow cytometric analysis of DNA and for culture, using transillumination-assisted microdissection. Precise stages have been identified by phase contrast microscopy of live cell squashes from adjacent segments. Each stage of the cycle showed a characteristic flow cytometric pattern with haploid (1C), diploid (2C) and tetraploid (4C) peaks. Stages I to VIII of the cycle showed an additional hypofluorescent (0.25-0.70C) peak due to a reduced dye-binding capacity of maturation phase-spermatids at steps 15 through 19. The appearance of the hypofluorescent haploid peak coincided with the second nucleoprotein transition at step 15 of spermiogenesis and the homogeneous condensation of the chromatin seen in electron microscopy. As a concomitant of the formation of disulphide bonds during epididymal maturation, the fluorescence intensity decreased further to reach a relative value of 0.07C in the cauda epididymidis. The constant 1C peak was raised by round and elongating spermatids (steps 1-14), 2C by spermatogonia, secondary spermatocytes and Sertoli cells, and the 4C peak by primary spermatocytes and spermatogonia at G2 or M phase of the mitotic cycle. The proportion of each peak accurately reflected the relative proportion of cells in most stages of the cycle when compared with morphometric measurements of histologic preparations. DNA flow cytometry is a suitable method for quantitative evaluation of cultured seminiferous tubule segment DNA. Although the relative yield of the meiotic reductive divisions in vitro is comparable with that observed in vivo, steps 9 and 15 of spermiogenesis involving nucleoprotein transitions and spermiation itself did not occur under the present culture conditions.

Animals↗
PubMed2005

Effect of protein kinase C alpha, caspase-3, and survivin on apoptosis of oral cancer cells induced by staurosporine.

AIM: To elucidate inhibition of protein kinase C alpha (PKC alpha) activity by staurosporine on apoptosis of oral cancer cell line tongue squamous cell carcinoma (TSCCa) cells and to clarify the role of survivin and caspase-3 in mediating apoptosis. METHODS: TSCCa cell viability was measured by MTT assay after 100 nmol/L staurosporine treatment. Apoptotic cells were identified by using phase contrast microscopy, acridine orange/ethidium bromide staining, and flow cytometry. Level of PKC alpha and its subcellular location were investigated using Western blot analysis. Expression of survivin and caspase-3 were evaluated using immunocytochemistry. RESULTS: Staurosporine significantly inhibited the cell viability of TSCCa cells in a dose- and time-dependent manner. Marked cell accumulation in G2/M phase was observed after 100 nmol/L staurosporine exposure for 6 h and 12 h. In addition, the percentage of apoptosis increased in a time-dependent manner, from 2.9% in control cultures to approximately 27.4% at 100 nmol/L staurosporine treatment for 24 h. Staurosporine displayed difference in inhibitory efficacy between cytosolic and membrance-derived PKC alpha. The content of PKCalpha in membrane versus cytosol decreased quickly, from 0.45 in ethanol-treated control cultures to 0.18 after staurosporine exposure for 24 h (P<0.01). After treatment with staurosporine, a time-dependent reduction of survivin and an activation of caspase-3 were observed in TSCCa cells. CONCLUSION: Staurosporine inhibited cell viability and promoted apoptosis in TSCCa cells. Inhibition of PKCalpha activity might be a potential mechanism for staurosporine to induce apoptosis in this cell line. The cleavage of survivin and activation of caspase-3 signaling pathway might contribute to PKC alpha inhibition-induced apoptosis.

Apoptosis↗
PubMed2005

In vivo platelet production from mature megakaryocytes: does platelet release occur via proplatelets?

Although platelets are universally accepted to be born from megakaryocytes (MKs), the mechanism by which platelets are formed and released from MKs in vivo remains controversial. One theory, known as the proplatelet theory, postulates that platelets are released from proplatelet processes protruding from MKs into sinusoids located in the bone marrow hematopoietic compartment. Proplatelet formation (PPF) has been observed in in vitro experiments involving detailed analyses of related molecular events. PPF has also been used as a marker of MK maturation. However, PPF is suggested to be a nonphysiological phenomenon. On the other hand, transmission electron microscopy (TEM) analyses have revealed platelet formation via explosive fragmentation of MK cytoplasm in bone marrow and lung capillaries prepared by immersion fixation. Moreover, TEM and scanning electron microscopy studies of liquid-cultured MKs kept in suspension show that platelet formation occurs without PPF. Rather, an explosive and global fragmentation of the MK cytoplasm composed of platelet territories has been reported as the mechanism of platelet formation. In addition, in vivo and ex vivo observations of platelet release from MKs with phase-contrast microscopy strongly support the explosive-fragmentation theory. With all observations taken into account, PPF may not be a prerequisite for platelet release from MKs under real-life conditions. In this review, a new "protoplatelet" concept is proposed to support the explosive-fragmentation theory. Additionally, the role of the lungs in platelet production is reviewed and discussed.

Animals↗
PubMed1995

Localization of nerves adjacent to goblet cells in rat conjunctiva.

Neural stimulation of the cornea induces conjunctival goblet cell mucous secretion. Immunofluorescence microscopy was used to determine if nerves are present near conjunctival goblet cells and what types of nerves are present. In euthanized rats, the local anesthetic lidocaine (1%) was placed topically on the ocular surface for 10 min to prevent goblet cell mucous secretion. The ocular surface tissues were removed and either fixed in formaldehyde and then frozen, or frozen first and then post-fixed in formaldehyde. Tissue was sectioned and nerves localized by indirect immunofluorescence microscopy, using antibodies to synaptophysin (indicates nerve, independent of type), vasoactive intestinal peptide (VIP, indicates parasympathetic nerves), tyrosine hydroxylase (TH, indicates sympathetic nerves), dopamine beta-hydroxylase (DBH, indicates sympathetic nerves), phenylethanolamine-N-methyltransferase (PNMT, indicates sympathetic nerves), and calcitonin gene-related peptide (CGRP, indicates sensory nerves). Goblet cells were identified by phase-contrast microscopy. Synpatophysin-containing nerves were present in the basolateral region of conjunctival goblet cells clusters. Nerve fibers immunoreactive to VIP were found in the conjunctiva along the epithelial-stromal junction and around the basolateral aspect of goblet cell clusters. Nerve fibers immunoreactive to TH and DBH were detected surrounding goblet cells and in the conjunctival stroma. Nerve fibers immunoreactive to CGRP were detected in the epithelium and at the epithelial stromal junction, but were not localized near goblet cell clusters. CGRP-containing nerve fibers were also detected in the conjunctival stroma under the epithelium. We conclude that efferent parasympathetic and sympathetic, but not afferent sensory, nerves appear to be located adjacent to conjunctival goblet cell clusters. Activation of efferent parasympathetic and sympathetic nerves could directly stimulate conjunctival goblet cell mucous secretion. Antidromic activation of afferent sensory nerves releasing neurotransmitters could stimulate goblet cell secretion by a paracrine mechanism.

Animals↗
PubMed1990

[Effect of retinoic acid activated mouse macrophage on human alveolar cell carcinoma of lung(A549) in vitro].

The tumoricidal activity of mouse macrophage (M phi) activated with retinoic acid (RA) and the effect of RA in combination with CP on mouse M phi were studied with phase contrast microscopy, light microscopy, and 3HTdR labelling technique. The results of our studies suggest that: (1) RA can activate mouse peritoneal M phi both in vivo and in vitro, and the RA-activated M phi are cytostatic and cytolytic to A549 cells. The degree of activation is related to the concentration or dosage of RA given, and (2) RA can enhance the function of CP-activated M phi. A summation effect was obtained by giving RA in combination with CP. This effect was related to the dosage of RA. When optimal dosage was given in combination with CP, a synergistic action in activating M phi could be obtained. Morphological changes could be seen under phase contrast microscope and light microscope in killed tumor cells.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed2003

Adhesive interactions between CD34(+)-derived dendritic cell precursors and dermal microvascular endothelial cells studied by scanning electron microscopy.

Dendritic cells are migratory cells. Before they extravasate from the circulation into the skin across capillary blood vessel walls, they have to interact with endothelial cells. Using a fluorimetric adhesion assay, we have recently shown that CD34(+)-derived dendritic cell precursors are able to bind to resting and stimulated dermal microvascular endothelial cells. In the present study, we attempted to visualize this process at an ultrastructural level. CD34(+) progenitor cells were purified from human cord blood samples by means of immunomagnetic beads, and dendritic cells were generated by culture in the presence of GM-CSF, TNF-alpha and hSCF for 5 days. Immature CD83(-) CD86(low) dendritic cells were added to human dermal microvascular endothelial cells grown to confluence on membrane chambers. After 2 h, unbound dendritic cell precursors were removed, and bound cells were prepared for routine scanning electron microscopy. We found that (1). dendritic cell precursors firmly adhere to microvascular endothelial cells, enveloping them with their surface processes; (2). dendritic cell precursors are extremely deformable as they squeeze through the dense network of microvascular endothelial cells; (3). microvascular endothelial cells form, in part, a multi-layered network rather than the typical cobblestone pattern as seen by phase-contrast microscopy. The morphology of dendritic cell precursors and of human dermal microvascular endothelial cells was examined here, for the first time, by scanning electron microscopy. These data further emphasize that CD34(+)-derived dendritic cells efficiently adhere to dermal microvascular endothelial cells.

Antigens, CD34↗
PubMed1962

Cinemicrophotographic observations on granule lysis in polymorphonuclear leucocytes during phagocytosis.

Phagocytosis of yeast cell walls and of Bacillus megaterium by human, rabbit, and chicken polymorphonuclear leucocytes has been observed by phase contrast microscopy and recorded on motion picture film. In suitably thin preparations intracellular events could be visualized well. Lysis of cytoplasmic granules began early in the course of the ingestion process, rupture occurring only in granules adjacent to the microorganism being engulfed. Formation of a visible vacuole about the ingested particle frequently followed degranulation. Chicken polymorphonuclear leucocytes, with their large phase-dense granules, were particularly suitable subjects for observations on detailed morphologic aspects of granule lysis. Rupture took place rapidly (0.1 second or less); in place of the granule there appeared a clear zone, often with a small phase-dense round structure in its center. Also accompanying granule lysis was an increase in phase density of the adjacent surface of the microorganism. Over the course of the following few seconds the darkening on the organism faded, the dense small body disappeared from view, and the clear zone contracted towards the engulfed particle. The observations are discussed in relation to the hypothesis that fusion takes place between the granule membrane and the invaginated cell membrane overlying the ingested particle, with discharge of granule contents directly into the phagocytic vacuole.

Animals↗
PubMed1990

Characterization of airborne dust in a soft paper mill.

The characteristics of airborne dust in a soft paper production plant have been characterized by scanning and transmission electron microscopy. A combination of X-ray diffraction, electron diffraction and energy-dispersive X-ray flouresence spectroscopy (EDX) was used to determine the structure and composition of the different components. Size distribution determination and phase identification were carried out. Besides the cellulose fibres, fibres of kaolinite, wollastonite, talc and other silicates were also identified. Gravimetric analysis and fibre counting by optical phase contrast microscopy were used to determine total dust and fibre concentrations. Total dust exposure at the plant was generally below 3 mg m-3. The respirable fraction of the total dust concentrations varied from 15 to 70%. The inorganic dust was 36 +/- 15% of the total dust. The ratio of inorganic fibres to total fibre concentration at the plant varied between 10 and 15%.

Air Pollutants, Occupational↗
PubMed1985

Studies of otoconia in the developing chick by polarized light microscopy.

Using polarized light microscopy we were able to observe the mineralization patterns of embryonic and neonatal chick otoconia. We compared preparations of freshly dissected material spread under mineral oil to material that had been treated with various fixatives and dehydration agents. We found that the standard fixation agent, glutaraldehyde, and some immersion oils etched embryonic chick otoconia but that fixation with 70% acetone or 70% alcohol followed by dehydration to 100% acetone or 100% alcohol left the otoconia intact. The size and shape of freshly dissected chick otoconia observed with polarized light microscopy were similar to those of acetone-fixed, critical-point-dried material examined by SEM. Embryonic forms of otoconia were found to have a fluted pattern that was different in morphology from otoconia found in hatched chicks and adults. Embryonic chick otoconia did not exhibit the multifaceted surface morphology seen in embryonic rat otoconia. Comparisons of the same fields of otoconia under phase contrast microscopy and polarized light microscopy indicated that the freshly dissected otoconia of embryos exhibit little or no unmineralized (non-birefringent) material but that glutaraldehyde-fixed otoconia exhibited unmineralized areas where etching had occurred. Size frequency distributions of freshly dissected embryonic and mature otoconia in five ages of embryos and hatched chicks were consistent with a hypothesized developmental sequence of otoconia.

Animals↗
PubMed1978

Liposomes from ionic, single-chain amphiphiles.

In studies of the minimum physiochemical requirements for lipid membrane formation, we have made liposomes from dilute, aqueous dispersions of C(8)-C(18) single-chain amphiphiles. In general, membrane formation from ionic soaps and detergents requires the presence of uncharged amphiphiles. Vesicles were characterized by phase-contrast microscopy, by trapping of ionic dyes, as well as by negativestain and freez-frature electron microscopy. They were typically heterogeneous in size, but the average diameter could be experimentally varied in some cases over the range of 1 to 100 micrometer. Uni-, oligo-, and multilamellar vesicles were observed. Membrane permeability to various solutes was determined in part by a new technique which utilized phase-contract microscopy; when impermeable vesciles exclude added solutes such as sucrose, refractive index differences are created between vesicle contents and surrounding medium, so that the vesicles appear bright in the phase microscope. Permeant solutes do not produce this effect. Spectrophotometric permeability determinations confirmed the results of this technique and provided quantitative measures of permeability. Monoalkyl liposomes have potential uses as models of biomembranes and in drug delivery. They are also relevant to the prebiotic origin of biomembranes.

Fatty Acids, Nonesterified↗
PubMed1990

GDA-J/F3 monoclonal antibody as a novel probe for the human sperm tail fibrous sheath and its anomalies.

GDA-J/F3 monoclonal antibody (MoAb) recognized an antigen in the fibrous sheath (FS) of human spermatozoa. This was based on: (i) intracellular localization of the antigen which was limited to the principal piece of the sperm tail; (ii) its absence from the cilia of trachea and nasal mucosa which lack FS; (iii) immunogold electron microscopy (IEM) which confirmed its ultrastructural localization to the FS. The antibody was used for the detection of abnormal germ cells in human semen. Nucleated cells other than spermatozoa (NCOS) obtained from oligozoospermic donors were screened with GDA-J/F3 MoAb using the indirect immunofluorescence test. The antibody which stains only the tails in normal cells, produced diffuse cytoplasmic immunofluorescence inside some spermatids. Using phase contrast microscopy, the tails in these spermatids were either present or undetectable. Electron microscopy studies of the NCOS showed the lack of the FS in those which had the tails (afibrous tail), or the presence of disorganized tails (tail dysgenesis) in the others. This antibody therefore provides a useful analytical tool for probing the FS as well as for the easy identification of certain abnormal germ cells in human semen.

Antibodies, Monoclonal↗
PubMed2004

Growth arrest and apoptosis of human hepatocellular carcinoma cells induced by hexamethylene bisacetamide.

AIM: To investigate the cellular effects of hybrid polar compound hexamethylene bisacetamide (HMBA) on the growth and apoptosis of human hepatocellular carcinoma cells and to provide the molecular mechanism for potential application of HMBA in the treatment of liver cancer. METHODS: Effects of HMBA on the growth of human hepatocellular carcinoma SMMC-7721 cells were assayed by MTT chronometry. Apoptosis induced by HMBA was detected by phase-contrast microscopy, flow cytometry, propidium iodide staining and immunocytochemical analysis. RESULTS: The growth of SMMC-7721 cells was significantly inhibited by HMBA, and the growth inhibitory rate was 51.1%, 62.6%, 68.7% and 73.9% respectively after treatment with 5.0, 7.5, 10.0 and 12.5 mmol/L of HMBA. In the cells treated with 10 mmol/L of HMBA for 72 h, the population of cells at sub-G(1) phase significantly increased, and the apoptotic bodies and condensed nuclei were detected. Moreover, treatment of SMMC-7721 cells with 10 mmol/L of HMBA down-regulated the expression of Bcl-2 anti-apoptotic protein, while slightly up-regulated the level of pro-apoptotic protein Bax. CONCLUSION: Treatment with 10.0 mmol/L of HMBA can significantly inhibit the growth and induce apoptosis of human hepatocellular carcinoma SMMC-7721 cells by decreasing the ratio of Bcl-2 to Bax.

Acetamides↗
PubMed1989

Statistical analysis of data on airborne asbestos levels collected in an EPA survey of public buildings.

Data from a recent EPA study of airborne asbestos levels in 49 buildings occupied by the General Services Administration (GSA) are statistically analyzed. The study focuses on differences between indoor and outdoor levels, and on differences among buildings with no asbestos-containing material (ACM) (6 buildings), buildings with ACM generally in good condition (6 buildings), and buildings containing damaged ACM (37 buildings). Seven indoor samples and a single outdoor sample were collected from most buildings and analyzed by TEM (transmission electron microscopy) using a direct preparation method. No statistically significant differences were detected in asbestos levels between indoors and outdoors or among the three categories of buildings. The average indoor concentration of asbestos was 0.00073 fibers/cc (f/cc) for all fibers and 0.00007 f/cc for fibers 5 microns or longer. The current OSHA occupational standard is 0.2 f/cc for fibers 5 microns or longer as measured by phase-contrast microscopy.

Air Pollutants, Occupational↗