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Coherent soft-x-ray radiation up to photon energies of 700 eV is obtained by focusing several-mJ, 10-fs near infrared laser pulses into a He gas jet. The observed nearly constant photon yield over several hundred eVs may be attributed to nonadiabatic self-phase matching, originating from a substantial ionization within a fraction of the optical cycle of the driving laser pulse.
We have devised, in guinea pigs, an improved ATPase technique which enables one to proceed from light to electron microscope study while preserving, on the ultrastructural level, the various membranous structures, in particular the Langerhans cell (LC) granules. Using this method, we have been able to confirm the action of acute, low-dose UVB on the surface enzymatic marker, ATPase. Moreover, this study has shown that the ATPase-negative LC contain abnormal LC granules or, more often, are deficient in LC granules. In a previous work, we have shown that, after epicutaneous application of a hapten, one successively observes an extensive adsorptive pinocytosis process, the disappearance of the membranous ATPase system, and the appearance of LC granules in the cytoplasm. Therefore we may suppose that, after UVB irradiation, the disappearance of the ATPase system and/or the possible alteration of the adsorptive pinocytosis process interrupts or alters the formation of LC granules. These successive events might play a vital role in the formation of the hapten--carrier protein-Ia antigen complex. In their absence in a large number of LC, following UV irradiation, epicutaneous application of a hapten would lead to the development of a state of immune tolerance.
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To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air-liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm2 x 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo.
The primate cornea was exposed to 300 nm UVR with five levels of radiant exposure from 0.08 to 0.6 Jcm-2. All cellular layers of the cornea were damaged at the 0.08 Jcm-2 exposure, and damage became more severe as the exposure level was increased. The corneal cells showed variable response in that essentially normal cells were found among damaged cells. Eight days post-exposure using the 0.6 Jcm-2 level, the epithelium had regained its normal thickness and was populated largely by normal appearing cells; however, the stroma showed damaged keratocytes and the loss of keratocytes. The corneal basement membranes (the epithelial basement membrane and the posterior limiting lamina) and the anterior limiting lamina were not damaged at any exposure level except for an isolated area along the epithelial basement membrane in one cornea. Therefore, one is lead to conclude that basement membranes are unaffected by UVR. The endothelium continued to demonstrate the loss of mitochondria, endoplasmic reticulum and some vacuoles at 8 days after exposure. However, the endothelium appeared to have resumed its physiological function as demonstrated by the reduced stromal oedema. This research gives the first complete description of UV-B induced corneal damage and repair of the full, in-depth cornea of the primate using the EM.
1. Ca2+ liberation induced in Xenopus oocytes by a poorly metabolized derivative of inositol 1,4,5-trisphosphate (3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate; 3-F-InsP3) was visualized using a video-rate confocal microscope to image fluorescence signals reported by the indicator dye calcium green-1. 2. Low (10-30 nM) intracellular concentrations of 3-F-InsP3 evoked Ca2+ release as localized transient 'puffs'. Progressively higher concentrations (30-60 nM) gave rise to abortive Ca2+ waves triggered by puffs, and then (> 60 nM) to a sustained elevation of Ca2+ followed by the appearance of propagating Ca2+ waves. At concentrations up to that giving waves, the frequency of puffs increased as about the third power of [InsP3], whereas their amplitudes increased only slightly. 3. The rise of cytosolic Ca2+ during a puff began abruptly, and peaked within about 50 ms. The peak free Ca2+ level was about 180 nM, and the total amount of Ca2+ liberated was several attomoles (10(-18) mol), too much to be accounted for by opening of a single InsP3-gated channel. The subsequent decline of Ca2+ occurred over a few hundred milliseconds, determined largely by diffusion of Ca2+ away from the release site, rather than by resequestration. Lateral spread of Ca2+ was restricted to a few micrometres, consistent with an effective diffusion coefficient for Ca2+ ions of about 27 microns2 s-1. 4. The peak amplitudes of puffs recorded at a given site were distributed in a roughly Gaussian manner, and a small proportion of sites consistently gave puffs much larger than the main population. Intervals between successive puffs at a single site were exponentially distributed, except for a progressive fall-off in puffs seen at intervals shorter than about 10 s. Thus, triggering of puffs appeared to be stochastically determined after recovery from a refractory period. 5. There was little correlation between the occurrence of puffs at sites more than a few micrometres apart, indicating that puff sites can function autonomously, but closely (ca 2 microns) adjacent sites showed highly correlated behaviour. 6. Puffs arose from sites-present at a density of about 1 per 30 microns2 in the animal hemisphere, located within a narrow band about 5-7 microns below the plasma membrane. 7. We conclude that Ca2+ puffs represent a 'quantal' unit of InsP3-evoked Ca2+ liberation, which may arise because local regenerative feedback by cytosolic Ca2+ ions causes the concerted opening of several closely clustered InsP3 receptor channels.
A photoinduced method for converting large quantities of silver nanospheres into triangular nanoprisms is reported. The photo-process has been characterized by time-dependent ultraviolet-visible spectroscopy and transmission electron microscopy, allowing for the observation of several key intermediates in and characteristics of the conversion process. This light-driven process results in a colloid with distinctive optical properties that directly relate to the nanoprism shape of the particles. Theoretical calculations coupled with experimental observations allow for the assignment of the nanoprism plasmon bands and for the first identification of two distinct quadrupole plasmon resonances for a nanoparticle. Unlike the spherical particles they are derived from that Rayleigh light-scatter in the blue, these nanoprisms exhibit scattering in the red, which could be useful in developing multicolor diagnostic labels on the basis not only of nanoparticle composition and size but also of shape.
Group A streptococci (Streptococcus pyogenes) of several different serological types were grown in fluorescein-labeled homologous type or group-specific globulins, thereby labeling the antigen-containing cell walls. Specific precipitation or inhibition of the labeled antibody, followed by continued incubation and examination at intervals by ultraviolet, phase, and dark-field microscopy, showed that new cell wall was then nonfluorescent. These nonfluorescent portions were differentiated by a reverse technique of culture in unlabeled globulin, followed by antibody precipitation, further growth, and fluorescent-antibody staining. This technique of differential labeling of cell wall has permitted following, for the first time in a living system, the fate of cell wall formed at different times. The results suggest that cell wall synthesis in actively growing cultures usually occurs simultaneously at at least two sites per coccus, each site representing stages in successive divisions, and that cell wall growth in Streptococcus pyogenes is not by diffuse intercalation with old wall, but is initiated at and extends both peripherally and centripetally from the coccal equator.
The penetration of ampicillin and ciprofloxacin through biofilms formed by Klebsiella pneumoniae was confirmed by transmission electron microscopic observation of antibiotic-affected cells at the distal edge of the biofilm. Because the bacteria nevertheless survived antibiotic treatment, some protective mechanism other than inadequate penetration must have been at work in the biofilm.
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Dramatic changes in cellular dynamics characterise the apoptotic execution phase, culminating in fragmentation into membrane-bound apoptotic bodies. Previous evidence suggests that actin-myosin plays a dominant role in apoptotic cellular remodelling, whereas all other cytoskeletal elements dismantle. We have used fixed cells and live-cell imaging to confirm that interphase microtubules rapidly depolymerise at the start of the execution phase. Around this time, pericentriolar components (pericentrin, ninein and gamma-tubulin) are lost from the centrosomal region. Subsequently, however, extensive non-centrosomal bundles of densely packed, dynamic microtubules rapidly assemble throughout the cytoplasm in all cell lines tested. These microtubules have an important role in the peripheral relocation of chromatin in the dying cell, because nocodazole treatment restricts the dispersal of condensed apoptotic chromatin into surface blebs, and causes the withdrawal of chromatin fragments back towards the cell centre. Importantly, nocodazole and taxol are both potent inhibitors of apoptotic fragmentation in A431 cells, implicating dynamic microtubules in apoptotic body formation. Live-cell-imaging studies indicate that fragmentation is accompanied by the extension of rigid microtubule-rich spikes that project through the cortex of the dying cell. These structures enhance interactions between apoptotic cells and phagocytes in vitro, by providing additional sites for attachment to neighbouring cells.
Photoelectron images of mammalian spermatozoa were obtained by subjecting the specimens to u.v.-irradiation and focussing the emitted electrons by electron optics (photoelectron microscopy). Guinea-pig, hamster and human spermatozoa were fixed in glutaraldehyde, deposited on conductive glass discs, and dehydrated. Sufficient quantities of photoelectrons were released from the surface of spermatozoa to produce images without staining, coating or metal shadowing. The large planar heads of guinea-pig spermatozoa were easily resolved with good delineation of acrosomal and postacrosomal regions. Residual vesicles could be visualized on the surface of the inner acrosomal membrane of spermatozoa that had undergone the acrosome reaction. Also detectable in these photoelectron images were finer membrane surface details, periodicities in the midpiece region of the tail which coincided with the distribution of mitochondria, and periodicities in the principal piece which appeared to be related to fibrous sheath components. Hamster spermatozoa were similarly well resolved but human spermatozoa were more difficult to image because of their increased surface curvature. The mechanism responsible for detection of these surface details is primarily topographical contrast rather than material contrast, since spermatozoa coated with a thin layer of gold or platinum exhibited similar features, although at reduced resolution, as the uncoated specimens.
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A quick embedding method employing UV polymerization reactions has been devised for embedding fibers in acrylic and methacrylate media. The resultant thin, flat embeddings are suitable for both light and electron microscopy.
The effect of UV-light on Pityrosporum yeasts (P.) was studied: P. yeasts cultured from the skin were spread on Dixon plates and irradiated with different UVB-and UVA-light dosages and read after three days, controls were not irradiated. Also P. yeasts, immediately after irradiation, were isolated from the plates and studied with an electron microscope. A significant growth inhibition or no growth at all was seen after 25, 50, 75 J/cm2 UVA and 900 mJ/cm2 UVB, a moderate inhibition after irradiation with 250 mJ/cm2 UVB. The growth inhibition was paralleled by ultrastructural degenerative alterations: clumping of ribosomes and lysis of nuclei. The amount of "stacked material" in the vacuoles was diminished or they were completely empty, the cell wall remained unchanged. Our results imply that the positive effect of sunlight on seborrhoeic dermatitis may well be explained by the direct influence of UV-light on the P. yeasts.
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