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  • 1
    ISSN: 0741-0581
    Keywords: Fluorescence microscopy techniques ; Poleward chromosome movement ; Microtubule dynamics ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Fluorescence microscopy techniques have become important tools in mitosis research. The well-known disadvantages of fluorescence microscopy, rapid bleaching, phototoxicity and out-of-focus contributions blurring the in-focus image are obstacles which still need to be overcome. Confocal fluorescence microscopy has the potential to improve our capabilities of analyzing cells, because of its excellent depth-discrimination and image processing power. We have been using a confocal fluorescence microscope for the study of the mechanism of poleward chromosome movement, and report here (1) a cell preparation technique, which allows labeling of fixation sensitive spindle antigens with acceptable microtubule preservation; (2) the use of image processing methods to represent the spatial distribution of various labeled elements in pseudocolour; (3) a novel immunoelectron microscopic labeling method for microtubules, which allows the visualization of their distribution in semithin sections at low magnification; and (4) a first attempt to study microtubule dynamics with a confocal fluorescence microscope in living cells, microinjected with rhodamine labeled tubulin.Our experience indicates that confocal fluorescence microscopy provides real advantages for the study of spatial colocalization of antigens in the mitotic spindle. It does not, however, overcome the basic limits of resolution of the light microscope. Therefore, it has been necessary to use an electron microscopic method. Our preliminary results with living cells show that it is possible to visualize the entire microtubule network in stereo, but that the sensitivity of the instrument is still too low to perform dynamic time studies. It will be worthwhile to further develop this new type of optical instrumentation and explore its usefulness on both fixed and living cells.
    Additional Material: 7 Ill.
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  • 2
    ISSN: 0741-0581
    Keywords: Proteins ; Molecular structure ; Negative-stain electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: The structure of ornithine decarboxylase (Mr ≍ 1.04 × 106) from Lactobacillus 30a was investigated by electron microscopy and x-ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod-shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod-shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted ± 45°. The two edge views were related by a 30° rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x-ray crystallographic analysis. A 4.0-Å resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10° with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x-ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy.
    Additional Material: 5 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Developmental Dynamics 204 (1995), S. 316-322 
    ISSN: 1058-8388
    Keywords: Helix-loop-helix ; Mesoderm ; Neural crest ; Mesenchyme ; Muscle ; Cartilage ; Heart valves ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The murine homologue of the Drosophila twist gene has been shown to be essential for head mesenchyme formation and to act as an inhibitor of muscle differentiation. This paper presents a detailed analysis of M-twist expression patterns from day 7 post coitum (p.c.) to day 18 p.c., indicating a more general function of the M-twist gene. At day 7 p.c., M-twist is expressed in the mesoderm outside the primitive streak. Later M-twist message is predominantly found in the somites, the head mesenchyme, the branchial arches, the limbs, and in the mesenchyme underneath the epidermis. Beginning at day 8 p.c., M-twist is mainly expressed in undifferentiated cells committed to muscle and cartilage development: this exspression is consistent with a suggested role of M-twist in inhibiting overt muscle and cartilage differentiation. However, during organogenesis, M-twistt is expressed in several areas of mesenchyme-epithelia interactions, suggesting additional tissue specific functions. © 1995 wiley-Liss, Inc.
    Additional Material: 8 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 120 (1966), S. 233-265 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The epidermal setae and the spinules of the digital lamellae of anoline and gekkonid lizards are shed periodically along with the rest of the outer layer of the skin. These structures are developed within the lamellae prior to ecdysis. The setae are larger and more complicated than the spinules and begin their development first. The setae of Anolis start as aggregations of tonofibrils beneath the plasma membrane of the presumptive Oberhautchen cells. These cells are arranged in rows parallel to the surface, several cell layers beneath the alpha layer of the skin. The developing setae protrude into the clear layer cells as finger-like projections, with the tonofibrils longitudinally oriented in the direction of growth. About 100 setae are formed by each Oberhautchen cells in Anolis. In late development, the clear layer cells lose their cellular contents and when shed along with all distal cells, retain a template of the new setae or spinules. The spinules and setae are formed before the fibrous and alpha layers of the new skin. The fibrous layer, which occurs only on the ventral (outer) layer of the lamellae, and the Oberhautchen with its setae and spinules, is considered the beta layer. The alpha layer, which occurs adjacent to the fibrous layer on the ventral surface and adjacent to the Oberhautchen on the dorsal (inner) surface, is morphologically identical to that of mammalian α keratin. The shed lizard skin consists of the alpha and beta layers as well as the degenerating cells of the outer epidermal generation, and the clear layer. The clear layer that is shed shows the template of the new setae and spinules developed in the new skin layer. The separation of the new from the old skin occurs along the intercellular space between the clear layer cells and the new Oberhautchen. The alpha layer of the skin is not fully keratinized at shedding. The setae of the digital lamellae of lizards represent unique epidermal structures  -  intracellular keratinized microstructures.
    Additional Material: 5 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 166 (1980), S. 275-288 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Serial sections ranging from very young embryos to hatched juveniles and whole embryos of Scyliorhinus show that dentition and dermal skeleton belong to two independent secondary developmental fields that differ both developmentally and structurally. The development of the dentition starts very early, with a thickening of the ectoderm in the region of the mouth (stage 04), the invagination of the dental lamina (stage 18), and the formation of the germs of the first generation (stage 20). Tooth replacement movements start only near the end of embryogenesis (stage 35). Scale germs, on the other hand, first begin to form at stage 24. Scales erupt shortly before the animal hatches (stage 43). Only one scale generation is formed during embryogenesis. The forces which erupt the scales may come from fluid pressures in vacuoles of the fibrous layer of the dermis. Those which erupt the teeth probably also result from similar fluid pressures. The crown and upper part of the base of scales and teeth are formed by cells of the inner dental epithelium which are differentiated from the ectoderm. They are also formed by odontoblasts which are derived from the vascular layer of the dermis. However, the basal plates of scales and teeth containing the anchoring fibers are formed by osteoblasts, which are derived from the fibrous layer of the dermis.
    Additional Material: 7 Ill.
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  • 6
    ISSN: 0730-2312
    Keywords: electron microscopy ; plasma membrane ; lymphoma cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Plasma membrane was isolated from the mouse T lymphoma cell line WEHI-22 using four different methods of cell disruption followed by centrifugal fractionation. Disruption by nitrogen cavitation or by shearing with a cell pump produced plasma membrane vesicles of similar buoyant density (1.10 g/ml) and morphological appearance. Few C-type virus particles were present. Cell disruption with 2% Tween-40 produced membrane vesicles of similar morphology but lower density (1.09 g/ml). All of the above preparations resulted in vesicles with aggregated intramembranous particles after freeze fracture. Microvesiculation with a sublytic concentration of a lysophosphatidylcholine analog (ET-12-H) (0.0032% w/v) produced small membrane vesicles which could be isolated without differential centrifugation. However, these had a slightly higher density than vesicles prepared by cavitation or shearing and were co ntaminated by virus particles. Unlike the other preparations, vesicles prepared with ET-I2-H had dispersed intramembranous particles. The enzyme γ-glutamyl transferase was enriched from 20- to 45-fold in the membrane preparations and proved a suitable plasma membrane marker for these cells whose 5′-nucleotidase content is very low.
    Additional Material: 5 Ill.
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  • 7
    ISSN: 0730-2312
    Keywords: T cells ; aging ; IL-2 ; IL-4 ; IFNγ ; CD45RB ; 3G11 ; 6C10 ; CD44 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Splenocytes from young adult or old C57BL/6NNia mice were stimulated in vitro with the anti-CD3∊ mAb, 145-2C11, in either soluble (2C11s) or plate-bound (2C11i) form. In the young group, each mode of cell activation resulted in peak DNA synthesis at ∼48 h of culture; at this time point, the old group exhibited response levels to 2C11s or 2C11i that were ∼40% of those in the young group. However, in the presence of 2C11i, splenocytes from old donors showed a delayed peak response which approached the peak levels attained in the young group. To analyze the responsiveness of the CD4+ T cell subpopulation, this cell type was isolated from spleens of young or old mice and was stimulated in vitro with 2C11s or 2C11i, in the presence or absence of added accessory cells (T cell-depleted, irradiated splenocytes). The induction of DNA synthesis by 2C11s was accessory cell dependent, and the response in the old group were markedly reduced in comparison to those in the young group. In contrast, stimulation of DNA synthesis with 2C11i was relatively accessory cell independent, resulted in higher response levels in both age groups, and lessened the disparity between age groups. The analysis of IL-2 and IL-4 secretion by stimulated CD4+ cells revealed that, in response to 2C11s and accessory cells, only IL-2 accumulation was detectable and the levels in the young group were ∼10-fold higher than the IL-2 levels in the old group. However, stimulation of CD4+ cells with 2C11i and accessory cells yielded improved IL-2 production and a detectable IL-4 response in the old group, whereas the young group exhibited a response profile similar to that induced by 2C11s. Further analysis of the IL-2, IL-4, and IFNγ mRNA levels in 2C11i-stimulated CD4+ cells revealed that old donor cells accumulated similar levels of IL-2 transcripts, but higher levels of IL-4 and IFNγ transcripts, than young donor CD4+ cells. Finally, we analyzed splenic CD4+ cells for membrane expression of four molecules - 3G11, 6C10, CD45RB, and CD44 - thought to demarcate CD4+ cell subsets with restricted patterns of cytokine production. The CD4+ cell fraction of individual mice contained higher percentages of cell phenotypes associated with increased IL-4:IL-2 production ratios (i.e., 3G11lo, CD45RBlo) and with increased IFNγ synthesis (i.e., CD44hi). Taken together, these data show marked alterations in the CD4+ cell subset composition in old mice, detected at the levels of subset marker expression and profiles of cytokine production. Moreover, conclusions regarding CD4+ cell competency in old donors can differ depending on the choices of stimuli and readouts for cell function in the experimental design. Therefore, age-related differences in T cell reactivity in vitro may be partially explained by the shifts in the representation of individual CD4+ subsets, each with potentially unique activation requirements and functional attributes.
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  • 8
    ISSN: 0730-2312
    Keywords: collagen type X ; gene regulation ; calcium phosphate ; reporter gene ; transfection ; hypertrophic chondrocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Collagen type X is a short, network-forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5′-flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non-tissue-specific positive regulatory element was present in the first intron. Third, a cell-type-specific enhancer element active only in hypertrophic chondrocytes was located between -2.4 and -0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291-296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements. J. Cell. Biochem. 66:210-218, 1997. © 1997 Wiley-Liss, Inc.
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  • 9
    ISSN: 0730-2312
    Keywords: vitamin D analogues ; vitamin D receptor ; ligand binding ; limited protease digestion ; ligand-dependent gel shift assay ; gene regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear hormone 1α,25-dihydroxyvitamin D3 (VD) has important cell-regulatory functions but also a strong calcemic effect. Therefore, various VD analogues have been synthesized and screened for their biological profile. In order to gain more insight into the molecular basis of the high antiproliferative but low calcemic action of the VD analogue EB1089, we characterized this compound in comparison to five structurally related VD analogues. The activities of the six VD analogues in in vitro assays (limited protease digestion assays for determining interaction with monomeric vitamin D receptor (VDR), ligand-dependent gel shift assays for showing the increase of DNA binding of VDR-retinoid X receptor (RXR) heterodimers, and reporter gene assays on different types of VD response elements for demonstrating the efficacy in nuclear VD signalling) were found to represent their biological potency (antiproliferative effect on different malignant cell lines). In this series, EB1089 proved to be the most potent VD analogue; that is, every structural modification (20-epi configuration, cis-configuration at position C24, or changes at the ethyl groups at position C25) appeared to reduce the determined activities mediated through the VDR of these analogues. Moreover, the modifications of EB1089 resulted in a loss of VD response element selectivity, suggesting that this parameter is very critical for the biological profile of this VD analogue. J. Cell. Biochem. 71:340-350, 1998. © 1998 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    American Journal of Anatomy 145 (1976), S. 283-317 
    ISSN: 0002-9106
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The fine structure of differentiating ameloblasts was studied in the lower second molar of 1-week-old kittens after perfusion fixation with and without subsequent decalcification.The differentiation zone was divided into three phases. In Differentiation 1, ameloblasts are about 27 μm long and face an uninterrupted basal lamina. The predentin adjacent to the basal lamina contains a few collagen fibrils oriented mainly at right angles to the ameloblast surface. This specialized predentin forms a well-defined layer, up to 1.5 μm thick, referred to as the junctional layer. In Differentiation 2, ameloblast processes extend through the basal lamina and the thickness of the junctional layer. The processes consist of cytoplasmic sheets forming a honeycomb-like network. Dentin starts to calcify after process-formation is underway. Two distinct types of odontoblast processes, having different shapes and contents, come in contact with the ameloblasts and push into the ameloblastic layer. In Differentiation 3, stippled material appears in the extracellular spaces between ameloblasts. Later, stippled material-like substances appear in the predentin close to the ameloblast apex and close to odontoblast processes within the dentin. Ameloblasts now are up to 40 μm high. Enamel secretion starts in small circumscribed areas which gradually enlarge, leading to the disappearance of the ameloblast processes. These findings are compared with results obtained in other species, including man, and their possible functional significance is discussed.
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