摘要:

AbstractBackground: Brefeldin A (BFA), when added to the medium of cultured mammalian cells, induces a reversible block of secretion and disrupts the Golgi apparatus whereas Golgi enzyme markers appear to redistribute into the endoplasmic reticulum (ER). It has been shown in addition that in mammalian cells, BFA would prevent the assembly of coatomer proteins (COP) onto membranes by inhibiting the GTP‐dependent interaction of the ADP‐ribosylation factor (ARF) with such membranes. The purpose of the present study is to analyze, by stereoelectron microscopy, the structural modifications of Golgi elements and of the ER‐Golgi relationship in a BFA‐sensitive yeast mutant,S. cerevisiae erg6.Methods:S. cerevisiae erg6cells were placed in a medium containing 100 μg/ml BFA dissolved in 1% alcohol and collected after exposures of 0.5, 1.5, 5, 10, 15, 20, 30, and 70 min to the drug. Yeasts placed in a BFA‐free medium but containing 1% alcohol served as controls. After fixation in 2% glutaraldehyde, the cells were postfixed in reduced osmium and embedded in Epon. Then 0.08–0.2 μm thick sections stained with lead citrate were examined with the electron microscope. Photographs of the thicker sections, tilted at ± 15° from the 0° position of the goniometric stage, were used to prepare stereopairs from which the three‐dimensional configuration of the organelles was visualized. Since BFA is known to prevent the interaction of ARF with membranes, the phenotype of thearf1mutant deficient in this protein was also examined for comparative purposes.Results: In control cells, as in wild‐type strains, two types of Golgi elements were observed: small networks of fine tubules seen close and occasionally connected to ER cisternae and coarser tubular networks showing nodular distensions having a size comparable to that of secretion granules. The latter networks were considered as trans‐Golgi elements and the former as cis‐Golgi elements. Several networks of both types were distributed throughout the cytoplasm. At short time intervals (0.5–5 min) of BFA treatment, the trans‐Golgi elements disappeared from the cytoplasm, while the ER‐connected cis‐Golgi elements developed and formed large spheroidal masses frequently showing concentrically arranged fine tubular networks. Such spheroidal, cage‐like structures later disappeared, and after 30 min Golgi elements were no longer identifiable, while ER cisternae assumed pleomorphic configurations as the cells showed signs of degeneration.S. cerevisiae arf1mutants presented a phenotype similar to that of BFA‐treatedS. cerevisiae erg6.Conclusions: It is therefore concluded that soon after exposure to BFA there is, in this sensitive yeast mutant, a transitory hypertrophy of the ER‐connected cis‐Golgi network presumably resulting from a block at the exit end of this compartment. At longer time intervals (i.e., after 30 min) the Golgi elements are no longer formed, and the cells present signs of c

ISSN:0003-276X    

DOI:10.1002/ar.1092410102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBackground and Methods: In an effort to review ultrastructural features of cells in the kidney of a male rat, transmission electron microscopy was used to study ultrathin sections.Results: One light cell in a collecting tubule contained a 2.3‐μm long linear array of electron‐dense asymmetric structures in a granular zone of greater electron density than the general cytoplasm. This inclusion body could be interpreted to consist of a parallel array of 100–150‐nm × 24‐nm electron‐dense rodlets, or a parallel array of 100–150‐nm×67‐nm tubules. The inclusion showed no association with any cell organelle. The origin, chemical nature, frequency of occurrence, and functional significance of this inclusion are unknown.Conclusions: Although this inclusion body somewhat resembles previously described inclusions or granules, the differences in dimensions, frequency, and relation to other cell structures suggest it is a new observation. ©

ISSN:0003-276X    

DOI:10.1002/ar.1092410103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBackground: One of the characteristic features of the two types (α and β) of “mitochondria‐rich” (chloride) cells in the gill epithelium of freshwater fishes is the presence in their apical region of tubulovesicular structures. A further analysis of the ultrastructural features of these apical elements as well as that of their modifications under various living conditions should help to understand better the respective rǒle of both α and β cells in these conditions.Methods: Atlantic salmon (Salmo salar) maintained in fresh water as well as tilapia (Oreochromis niloticus) maintained either in fresh water or in deionized water or in 20% saltwater were examined. Measurements of surface areas of apical structures in the various living conditions were also performed.Results: In the α cells of freshwater fishes, the apical structures consisted of isolated vesicles containing a filamentous material resembling that coating the apical surface. They were closely related to the apical plasma membrane and did not penetrate the region containing the tubular system. When fishes were transferred to deionized water, the number of the apical membrane folds increased significantly, as did the number and size of apical structures which became elongated. In saltwater‐adapted fishes, the apical structures showed a tendency to collapse and took the appearance of flattened and slightly curved elements. These observations tended to indicate that in α cells the apical structures were extensions of the apical plasma membrane and thereby might be implicated in sodium uptake when fishes are placed in fresh or deionized water and in chloride excretion when they are transferred to salt water. In β cells, the apical structures were usually separated from the apical plasma membrane by a zone rich in cytoskeleton elements. They penetrated deeply into the supranuclear region, where they intermingled with the elements of the tubular system. They consisted mainly of tubular elements that contained a material resembling that present in the trans tubular Golgi network from which they might originate. The apical structures remained unaltered in β cells whatever the medium (fresh or deionized water) in which the fish was placed.Conclusions: The α cells which are usually thought to be mainly involved in chloride excretion when fishes are transferred into seawater might also be implicated in sodium uptake in freshwater living conditions. The rǒle of β cells, in contrast, still remains to be established. ©

ISSN:0003-276X    

DOI:10.1002/ar.1092410104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBackground: The problem of how the functional compartments of the Golgi apparatus organizes during cell differentiation to become a well‐formed Golgi apparatus is as yet an unresolved issue. This study was designed to define the involvement of the trans‐Golgi network (TGN) and the Golgi stack in organizing the Golgi apparatus.Methods: The distribution of the TGN marker enzyme was examined in the ameloblast of developing rat molar tooth germs using cytochemistry with Co‐enzyme A phosphatase (CoA Pase) and cytidine monophosphatase (CMPase).Results: Typically formed Golgi apparatus was observed in the secretory ameloblast but not in the presecretory ameloblast. Organization of the Golgi apparatus through the presecretory ameloblast was noted. In the presecretory ameloblast, Golgi stacks of different sizes and clusters of small vesicles were located in the cytoplasm lateral to the nucleus. The saccules with enzymes marked for TGN were also observed in the cytoplasm lateral to the nucleus. These saccules were adjacent to the cluster of small vesicles and/or the Golgi stack. Upon cell differentiation, Golgi stacks were seen in line along the long axis of the cell, and the file of the stacks in the cytoplasm lateral to the nucleus was formed. The positive saccule was seen in a parallel line equal to the length of the Golgi stacks.Conclusions: In organizing the Golgi apparatus, the development process of the TGN and the Golgi stack appear to be different, and new Golgi stacks seem to be formed through the accumulation of small vesicles near the pre‐existing TGN. © 1995 Wiley

ISSN:0003-276X    

DOI:10.1002/ar.1092410105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBackground: Previous studies of variation in cartilage characteristics with age have involved comparison of young and adult individuals, but no data on short‐term age‐related change are available. Such data are important for studies of the response of cartilage to experimental stimuli in young rabbits, to distinguish the response to the stimuli from accompanying age‐related changes.Methods: We used light microscopy to study the thickness, cell density, and degree of histological definition of articular cartilage on the femoral trochlea of 6‐, 7‐, and 8‐week‐old rabbits.Results: Thickness and cell density both decline significantly with age. The decline in cell density is more marked in surface layers of cartilage and is accompanied by an increase in the safranin O‐staining affinity of the extracellular matrix and an extension of this affinity towards the surface.Conclusions: Our results indicate that the synthesis of matrix components becomes more important relative to proliferative activity. The traditionally defined histological layers (zones I, II, III, and IV) are not clearly distinguishable in rabbits of this age. In 6‐ and 7‐week old animals only a “surface” (I/II) and a “deep” layer (III) can be distinguished. By 8 weeks, zones I and II are well defined but the mineralization front (marking the boundary between zones III and IV) is still abs

ISSN:0003-276X    

DOI:10.1002/ar.1092410106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBackground: The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact that the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position.Methods: The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1‐week‐old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6‐week‐old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2–3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures −30°C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti‐mouse IgM for demonstration of binding.Results: Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intercellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them.Conclusions: Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions. © 1995

ISSN:0003-276X    

DOI:10.1002/ar.1092410107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBackground: Cells interact with the extracellular matrix through a family of cell surface receptors known as integrias. Ligand specificity of a given integrin is determined in part by the type of α and the type of β subunit comprising it. Accumulating evidence suggests that integrinligand binding in some systems influences cell behavior through tyrosine phosphorylation of intracellular proteins.Methods: In this study, we utilized immunohistochemistry to examine the expression of β1and β4integrin subunits as well as tyrosine phosphorylation in normal keratinocytes and in keratinocytes migrating to form a wound epithelium. An adhesion assay was used to determine if freshly isolated keratinocytes could interact with fibronectin and collagen. Polyacrylamide gel electrophoresis followed by immunoblotting was employed to compare β1integrins in migrating and nonmigrating keratinocytes.Results: In normal epidermis, β1and β4localized primarily to basal cells, where both subunits were generally distributed over all parts of the cell periphery. Except for a modest presence in suprabasal cells and a minimal presence adjacent to the epidermal basement membrane, phosphotyrosine (ptyr) had a similar distribution. In migrating keratinocytes, β1, β4, and ptyr localized most heavily at the interface between the forming wound epithelium and the wound bed. Adhesion assays using keratinocytes from normal epidermis revealed a population of cells that could specifically adhere and spread on fibronectin and type I collagen. Immunoblots of β1subunits from normal and migrating keratinocytes showed no increase in amount of β1, nor did the apparent size of β1change in migrating compared to normal cells.Conclusions: The heavy accumulation of β1and β4at the wound bed interface in migrating cells suggests that these subunits may be involved in attachments of migrating cells to extracellular matrix proteins in the wound. The accumulation of ptyr in the same region further suggests that integrin‐ligand interaction in keratinocytes modulates cell behavior through phosphorylated proteins. The fact that freshly isolated newt keratinocytes could adhere and spread on fibronectin or collagen shows that these cells are constitutively activated. This view is supported by the absence of any evidence that the β1in migrating keratinocytes is larger and therefore more mature than β1in normal keratinocytes. By comparison, β1integrins on human keratinocytes are not constitutively activated (Takashima and Grinnell, 1985; Toda et al., 1987; Guo et al., 1990, 1991), a difference that may explain why epidermal wound healing is faster in newts than in humans. © 1

ISSN:0003-276X    

DOI:10.1002/ar.1092410108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBackground: Abundant actin filaments are present in myoid cells and Sertoli cells in the testis. In the adult rat, the filaments form a lattice arrangement within the myoid cell, and show a hexagonal pattern in the basal junctional regions of Sertoli cells.Methods: Isolated seminiferous tubules and frozen sections were prepared from juvenile to adult Wistar rat testes, stained with FITC‐conjugated phalloidin, and observed by confocal microscopy. Unilateral cryptorchidism was induced in adult rats, and seven days later, their testes were also examined.Results: In the myoid cell, parallel actin filaments running circularly around the seminiferous tubules were observed at 15 and 20 days of age. Then, at 30 days, actin filaments arranged longitudinally along the tubular long axis appeared in addition to the circular bundles. A lattice arrangement of actin‐filament bundles in myoid cells became obvious at 40 days, when elongated spermatids are found in the tubule. Actin filaments in the basal junctional regions of Sertoli cells did not acquire the hexagonal pattern seen in the adult testis until 30 days of age. In the cryptorchid testes, the arrangement of actin filaments in the both cells showed a remarkable change compared to the control testis; the filaments became thinner and disrupted.Conclusions: A lattice arrangement of the actin filaments in the myoid cell appear at around 30 days, before the completion of spermatogenesis. A hexagonal pattern of the filaments in the junctional regions of Sertoli cells has already developed at this age. Cryptorchidism affects the actin filaments of the both cells. © 1995 Wiley‐Lis

ISSN:0003-276X    

DOI:10.1002/ar.1092410109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBackground: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), we investigated the effects of these two cytokines on death of luteal cells in vitro.Methods: Mouse luteal cells were cultured in serum‐free medium with TNF‐α at 0,500,1,000,3,000, or 5,000 U/ml in the presence or absence of IFN‐γ at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3′ end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy.Results:On day 3 of culture, IFN‐γ alone at 1,000 U/ml or TNF‐α alone at 500–5,000 U/ml did not decrease the number of viable cells, but a combination of IFN‐γ (1,000 U/ml) and TNF‐α (5,000 U/ml) did. On day 6, IFN‐γ alone at 1,000 U/ml or TNF‐α alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF‐α alone at 5,000 U/ml did, and combinations of IFN‐γ and TNF‐α at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF‐α. On days 3–6 of culture, combinations of IFN‐γ and TNF‐α that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder‐like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis.Conclusions: The presence of IFN‐γ modulates the ability of TNF‐α to induce a reduction in the number of viable cells, although TNF‐α alone at high concentrations can

ISSN:0003-276X    

DOI:10.1002/ar.1092410110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBackground: Characteristic membrane changes in spermatozoa culminating in acrosome reaction and sperm‐egg fusion, and suspected involvement of actin‐containing cytoskeleton in membrane changes in general, prompted us to investigate subcellular distribution of actin and actin‐binding proteins in bovine spermatozoa subjected to various extractions which sequentially denude the sperm investments.Methods: Spermatozoa were treated with either 1% SDS, 0.1% Triton X‐100, 0.1% Hyamine, or 1 M MgCl2or were sonicated. Immunostaining of actin, α‐actinin, spectrin, and acrosin as well as electron microscopic analysis of extracted spermatozoa were carried out.Results: Extractions caused evagination of the acrosomal lamina which retained focal contacts with the inner acrosomal membrane. Extractions further revealed lateral prongs at the anterior border of the postacrosomal sheath. Labeling for α‐actinin and spectrin was localized in the acrosinpositive acrosomal lamina, neck, and principal piece, the latter containing also relatively extraction‐resistant oligomeric or polymerized actin. In the postacrosomal area, actin was accumulated in the extraction‐resistant posterior ring structure and anteriorly at the sites apparently related to the lateral prongs. Notably, spectrin reactivity was enhanced by MgCl2in head, neck, and principal piece, and sonication abolished cytoskeletal immunoreactivity in the head.Conclusions: Destabilization of membranes with selected extractions induces changes in the acrosomal lamina mimicking acrosomal vesicle formation. The lateral prongs and posterior ring structure, respectively, may serve as anterior and posterior anchors for the extraction‐resistant post‐acrosomal sheath. The lateral prongs may also be a merger zone for actin, α‐actinin, and spectrin with important implication on sperm function. The latter two proteins may be involved in acrosomal vesicle formation. It is apparent that extractions have a significant effect on the detectability of sperm cytoskeletal elements

ISSN:0003-276X    

DOI:10.1002/ar.1092410111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY