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PubMed1991

Detection of photorepair of UV-induced thymine dimers in human epidermis by immunofluorescence microscopy.

To investigate the effect of visible light on the level of UV-induced thymine dimers in human epidermal cells in vivo, we exposed volunteers to UV-B alone, or to a serial combination of UV-B and visible light. Dimers were assayed in skin sections by immunofluorescence microscopy with a monoclonal antibody against the cyclobutyl thymine dimer. The dimer-specific fluorescence from epidermal cell nuclei, identified by counterstaining with propidium iodide, was quantified through computer-mediated image processing and analysis. After a single UV exposure (2-3 MED), significant dimer-specific fluorescence was measured, but no difference could be detected between skin kept in the dark after UV-irradiation and that exposed to visible light. In three other experiments, the UV dose was split into 3 parts (1 MED each), given at 2.5-h intervals. Half of the skin area was exposed to visible light following each dose fraction. After the second and third dose fractions, skin areas treated with visible light clearly showed lower levels of dimers (i.e., about 40% reduced) than skin kept in the dark. The results provide evidence that photorepair of dimers does occur in human skin, but not immediately after a first UV exposure of naive skin.

Antibodies, Monoclonal↗
PubMed1993

The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration.

The ftsZ gene is essential for cell division in both Escherichia coli and Bacillus subtilis. In E. coli FtsZ forms a cytokinetic ring at the division site whose formation is under cell-cycle control. In addition, the FtsZ from E. coli has a GTPase activity that shows an unusual lag in vitro. In this study we show that FtsZ in Bacillus subtilis forms a ring that is at the tip of the invaginating septum. The FtsZ ring is dynamic since it is formed as division is initiated, changes diameter during septation, and disperses upon completion of septation. In vitro the purified FtsZ from B. subtilis exhibits a GTPase activity without a demonstrable lag, but the GTPase activity is markedly dependent upon the FtsZ concentration, suggesting that the FtsZ protein must oligomerize to express the GTPase activity.

Bacillus subtilis↗
PubMed1979

Ultrastructural and cell surface changes of human psoriatic skin following Goeckerman therapy.

Ultrastructural and cell surface studies of skin in psoriatic patients prior to and after Goeckerman therapy (crude coal tar and UVB-light) have demonstrated significant cellular changes following this treatment: hyperactivity of melanocytes with melanosome polymorphism, increase of desmosomes, tonofilaments, keratohyaline granules, a decrease in mitochondria, keratinosomes, polysomes, dark cells and a reduction in size of nuclei and nucleoli. The enlargement of intercellular spaces and the redundancy of basement membrane were also reduced. Langerhans cells were moderately decreased and exhibited a normal ultrastructural pattern. No significant changes in cutaneous nerve distribution or morphology were observed in these cases. Scanning electron microscopy following treatment revealed a regular surface and orientation of corneocytes, with flattened surfaces; and a reduction of their ridges on the surfaces, as well as of the intercellular spaces and red blood cells. These findings indicate that Goeckerman therapy restored the ultrastructural and cell surface pattern in the psoriatic skin by inducing orthokeratinogenesis, development of the tonofibrillar-desmosome system, and decrease in mitochondria, nuclei and nucleoli.

Coal Tar↗
PubMed1982

Ultrastructural changes in the nucleus of a human epithelial cell line exposed to cytotoxic agents. The effect of PUVA and cadmium.

An established human epithelial cell line was exposed to cytotoxic doses of photo-activated 8-methoxy psoralen (PUVA) and to cadmium (Cd++) and the cells' nuclear ultrastructure were compared with controls. PUVA and Cd++ have some common biological features. Both substances induced nuclear indentations and cytoplasmic invaginations, while only PUVA caused an increase in nuclear bodies. After cadmium exposure, clusters of large-sized perichromatin granules (PG) were seen as well as an increase in chromatin margination. After PUVA, an increase in singularly distributed PGs was observed. The nuclear envelope was not affected by PUVA while Cd caused extensive disruption of this membrane. A strain of the epithelial cell line was made resistant to 100 mumol Cd/l and its nuclear ultrastructure was examined. An increase in chromatin margination was seen compared with its parent line. When this Cd resistant cell strain was exposed to 200 mumol Cd/l no ultrastructural changes took place. An attempt to produce a PUVA resistant epithelial cell strain was unsuccessful. Some functional aspects of the different morphological parameters observed are discussed.

Cadmium↗
PubMed1975

Characterization of an in vitro persistent-state measles virus infection: establishment and virological characterization of the BGM/MV cell line.

The parameters of a persistent-state measles virus infection in BGM/MV cells were examined. The BGM/MV cell line was established by cocultivation of measles virus-infected primary C3H mouse brain cells with a stable line of African green monkey kidney cells (BGM). Initially, a morphologically mixed population of cells existed:BGM-like (epithelioid) and fibroblasts. Gradually the fibroblasts were replaced by BGM-like cells, resulting in a morphologically homogeneous population. Measles cytopathic effect was noted 2 days after initiation of this culture and persisted for approximately 290 days. The time of disappearance of viral cytopathic effect corresponded to the time at which morphological homogeneity was reached. Low titers of infectious measles virus were detected in the BGM/MV culture up to 20 days postseeding; thereafter none was observed. After 440 days in culture, 100% of BGM/MV cells demonstrated intractyoplasmic measles antigen by immunofluorescence. Nuclear fluorescence was never observed. Electron microscopy revealed the presence of measles virus mucleocapsid within the almost completely filling the cytoplasm of BGM/MV cells. The plasma membrane of these cells appeared normal; no maturing or budding particles were observed. Measles virus hemagglutinin was not detected in either clarified cell lysates or in supernatant culture fluids. Cell membrane alteration by measles virus was detected in less than 1% of these cells by hemadsorption and by membrane immunofluorescence. The hemadsorption activity of the cells could be enhanced (30 to 70%) by treatment with actinomycin D or enucleation with cytochalasin B; these treatments, however, were unsuccessful in inducing detectable levels of measles hemagglutinin. Treatment of BGM/MV cells with 5-bromo-2'-deoxyuridine (BUdR) at 5 to 50 mug/ml and cytosine arabinoside at 1 to 50 mug/ml failed to enhance hemadsorption activity. Doses of 5-bromo-2'-deoxyuridine ranging from 5 to 200 mug/ml and of actinomycin D ranging from 0.1 to 10 mug/ml were ineffective in inducing the synthesis of infectious virus. Various physical methods of induction of infectious virus was also unsuccessful.

Animals↗
PubMed1982

Virus-like particles and lytic plaque formation in lawns of Candida albicans.

The antifungal agent Aculeacin A at subinhibitory levels induced lytic plaques in lawns of Candida albicans. Electron microscopic examination of plaque lysates suspended in phosphotungstic acid revealed the presence of spherical particles 12, 18, and 28 to 30 nm in size. Particles were also found in ultrathin sections of treated C. albicans cells. The plaque lysate lost infectivity after treatment with UV light, heat treatment at 80 degrees C for 10 min, or being held at pH 2 for 30 min.

Antifungal Agents↗
PubMed1991

Salt-mediated multicell formation in Deinococcus radiodurans.

The highly radiation-resistant tetracoccal bacterium Deinococcus radiodurans exhibited a reversible multi-cell-form transition which depended on the NaCl concentration in the medium. In response to 0.8% NaCl addition into the medium, the pair/tetrad (designated 2/4) cells in a young culture grew and divided but did not separate and became 8-, 16-, and 32-cell units successively. In exponential growth phase, the cells divided in a 16/32 pattern. Potassium ions were equally effective as Na+ in mediating this multicell-formation effect; Mg2+, Li+, and Ca2+ also worked but produced less multiplicity. This effect appears to be species specific. This-section micrographs revealed that in a 16/32-cell unit, eight 2/4 cells were encased in an orderly manner within a large peripheral wall, showing five cycles of septation. Our results suggest the presence of a salt-sensitive mechanism for controlling cell separation in D. radiodurans.

Cell Division↗
PubMed1992

Chromosomal structure of the halophilic archaebacterium Halobacterium salinarium.

The chromosomal structure of the extremely halophilic archaebacterium Halobacterium salinarium was examined. Sheared chromosomes prepared from the bacteria in the late exponential phase were separated into two peaks (peaks I and II) by sucrose gradient centrifugation, suggesting that the chromosomes consist of two parts differing in quality. The UV spectra of peaks I and II resembled those of DNA and eukaryotic chromatin, respectively. Electron microscopic observations revealed that the major component of peak I was protein-free DNA, while the major components of peak II were rugged thick fibers with a diameter of 17 to 20 nm. The rugged fibers basically consisted of bacterial nucleosome-like structures composed of DNA and protein, as demonstrated in experiments with proteinase and nuclease digestion. Whole-mount electron microscopic observations of the chromosomes directly spread onto a water surface revealed a configuration in which the above-described regions were localized on a continuous DNA fiber. From these results it is concluded that the H. salinarium chromosome is composed of regions of protein-free DNA and DNA associated with nucleosome-like structures. Peaks I and II were predominant in the early exponential phase and stationary phase, respectively; therefore, the transition of the chromosome structure between non-protein-associated and protein-associated forms seems to be related to the bacterial growth phase.

Animals↗
PubMed2001

Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization.

Recently we reported an unusual multicellular organization in yeast that we termed stalk-like structures. These structures are tall (0.5 to 3 cm long) and narrow (1 to 3 mm in diameter). They are formed in response to UV radiation of cultures spread on high agar concentrations. Here we present an anatomical analysis of the stalks. Microscopic inspection of cross sections taken from stalks revealed that stalks are composed of an inner core in which cells are dense and vital and a layer of cells (four to six rows) that surrounds the core. This outer layer is physically separated from the core and contains many dead cells. The outer layer may form a protective shell for the core cells. Through electron microscopy analysis we observed three types of cells within the stalk population: (i) cells containing many unusual vesicles, which might be undergoing some kind of cell death; (ii) cells containing spores (usually one or two spores only); and (iii) familiar rounded cells. We suggest that stalk cells are not only spatially organized but may undergo processes that induce a certain degree of cell specialization. We also show that high agar concentration alone, although not sufficient to induce stalk formation, induces dramatic changes in a colony's morphology. Most striking among the agar effects is the induction of growth into the agar, forming peg-like structures. Colonies grown on 4% agar or higher are reminiscent of stalks in some aspects. The agar concentration effects are mediated in part by the Ras pathway and are related to the invasive-growth phenomenon.

Culture Media↗
PubMed1995

Microcapsules obtained from complex coacervation of collagen and chondroitin sulfate.

Microcapsules composed of collagen and chondroitin sulfate were obtained by complex coacervation and characterized by DSC, optical microscopy, SEM, and UV-Vis spectroscopy. Composition of the microcapsules could be adjusted by the feed ratio and the pH of the solution. Prepared under low temperature and aqueous solution, the process is most suitable for encapsulating delicate bioactive agents. Albumin as a model protein was encapsulated with a loading level of up to 95% by weight. Degradation rate of the microcapsules decreased with the concentration of the crosslinking agent glutaraldehyde and increased with the bacterial collagenase level. Correspondingly the release of albumin could also be varied by the cross-linking degree of the microcapsules.

Albumins↗
PubMed2003

A photoreactive fluorescent marker for identifying eosinophils and their cytoplasmic granules in tissues.

Here we describe a simple histochemical technique that provides an improved approach to identifying eosinophil components in tissues through the formation of photoreactive complexes that produce stable fluorescent emissions. This method worked readily with histological tissue sections 6-60 microm thick, which were fixed in neutral buffered formalin (NBF), and with cell suspensions similarly fixed and unfixed. Deep red (>605 nm) fluorescent emissions were produced by eosinophil-specific granules when exposed to broadband excitation spectra from a 100-W mercury lamp source (510-590 nm), as well as single-wavelength excitations from both an argon laser (488 nm) and a UV-visible laser (514 nm). The fluorophore-granule complex emissions increased in intensity during the first minute of continuous photoexcitation, then remained stable (>10 min). All nonspecific autofluorescence phenomena associated with these tissues were photobleached in the first minute, including areas of background Biebrich scarlet binding where photoreactive complexes were not formed (i.e., collagen), indicating environmental influences on the fluorophore. This technique allows the visualization of eosinophil granules over a greater period of time than is usually permissible with standard fluorescent markers. Therefore, techniques such as confocal microscopy can be utilized to their fullest extent, providing much more detailed information on the location and distribution of the cytoplasmic contents of eosinophils.

Animals↗
PubMed2006

An unexpected role for keratin 10 end domains in susceptibility to skin cancer.

Keratin 10 (K10) is a type I keratin that is expressed in post-mitotic suprabasal keratinocytes of the skin. Based on cell culture experiments and transgenic mouse studies, it has been proposed that K10 suppresses cell proliferation and tumor formation in the skin. Furthermore, the ability of K10 to suppress cell proliferation was mapped to its unique N- and C-terminal protein domains. In the present study, we modified the endogenous keratin 14 (K14) gene of mice using a knock-in approach to encode a chimeric keratin that consists of the K14 rod domain fused to the K10 head and tail domains (K1014chim). This transgene was expressed in the basal layer of the epidermis and the outer root sheath of hair follicles. Unexpectedly, we found that the K10 end domains had no effect on basal keratinocyte proliferation in vivo. Moreover, when subjected to a chemical skin carcinogenesis protocol, papilloma formation in mutant mice was accelerated instead of being inhibited. Our data suggest that the increased tumor susceptibility of K1014chim mice is in part due to a suppression of apoptosis in mutant keratinocytes. Our results support the notion that intermediate filaments, in addition to their function as cytoskeletal components, affect tumor susceptibility of epithelial cells.

Animals↗
PubMed2004

Determination of biologically active substances in roasted coffees using a diode-array HPLC system.

We studied the simultaneous quantitative analysis of biologically active substances, such as nicotinic acid, trigonelline, caffeine, qunolinic acid and tannic acid and pyrogallic acid, in several roasted coffees by an HPLC/diode-array system with a home-made sol-gel and ODS-2 columns. A simple method for simultaneous quantitative analysis of biologically active substances in the coffee brew became feasible by an HPLC/diode-array system with a sol-gel column at a single wavelength of 210 nm. The most efficient condition of the Rs value was above 1.05 when two sol-gel columns were connected. In addition, the elution behavior of nicotinic acid in brew extracted from commercially available coffee beans suggests the thermal decomposition process during roasting, and indicated the maximum value for full city roasted coffee.

Biological Factors↗
PubMed1995

A qualitative ultrastructural study of melanocytes after grenz-ray and UVA irradiation.

Seven female human subjects were irradiated with 4 Gy of grenz rays and 30J/cm2 of long-wave ultraviolet (UVA) radiation once a week for three weeks. 6/7 subjects when irradiated on the back developed a clearly visible pigmentation due to both grenz-ray and UVA pigmentation. The effect on epidermal melanocytes was observed with transmission electron microscopy. Ultrastructural changes in melanocytes following both grenz-ray and UVA irradiation were an increase in the number of premature and mature melanosomes, elongation and protrusion of cytoplasm, sometimes indented nuclei, and the development of multilamella of basal lamina. Compared with UVA-irradiated skin, in the same individuals the melanocytes seemed somewhat more hypertrophic after grenz-ray irradiation. In general the observed qualitative ultrastructural differences between UVA- and grenz-ray-irradiated melanocytes were limited, indicating that the influence of grenz-rays is similar to that of UVA.

Adult↗
PubMed2000

Effects of KrF excimer laser irradiation on human dental enamel.

Spectra of human dental enamel was recorded in the 200-400 nm UV region. It showed the weak band at 280 nm which were present in enamel protein. Excimer laser are gas lasers which emit light with photochemical decomposition. The wavelength depends on the 248 nm with krypton-fluoride. The enamel surfaces of extracted human teeth were exposed to KrF excimer laser by an energy density range from 29.6 to 3200 J/cm2. The purpose of this study was to investigate the changes with photo chemical reaction in enamel by light microscopy, SEM and X ray diffraction method. Results of analyses suggested that the observed changes of enamel exposed to this laser were the alpha and beta-tricalcium phosphate (TCP) phase in small amounts. No histological changes were observed in grain boundaries of cross sectioned lased enamel under light microscopy. The SEM examination revealed a roughened surface with bubble formation at 800-3200 J/cm2. SEM of etched enamel surface with 0.1 N HCl after laser irradiation at 400-800 J/cm2 showed the extension along the length of the rods. At 1600-3200 J/cm2, there appeared to be a melting of prism structures, because of conversion of photon energy into thermal energy. These results showed the KrF excimer laser irradiation to dental enamel might be a new type of treatment modality and diagnosis in preventive dentistry.

Calcium Phosphates↗
PubMed2005

Fabrication of a hydrogen peroxide biosensor based on a self-assemble composite oxide film.

A titania film with horseradish peroxidase (HRP) was immobilized on indium tin oxide (ITO) electrodes to act as a biosensor of hydrogen peroxide via the self-assembling and sol-gel route. The film was characterized by the atomic functional microscopy, electrochemical and UV-vis spectroscopy methods. The resulting biosensor exhibited fast amperometric response and good stability to hydrogen peroxide (H2O2). The linear range for H2O2 determination was from 2 x10(-6) to 1.45 x 10(-3) M, with a detection limit of 4 x10(-6) M based on S/N=3. The apparent Michaelis-Menten constant of the biosensor to H2O2 was estimated to be 1.5 mM, showing the HRP on the films had the higher biologic activities.

Biosensing Techniques↗