摘要:

The scanning electron microscope (SEM) has become a powerful tool for ultrastructural research with improvement of the instrument's resolution and progress in specimen preparation techniques. With regard to resolution, it has been improved step‐by‐step in this decade and, in 1985, an ultra‐high resolution SEM (UHS‐T1) was developed, with a resolution of 0.5 nm. Concerning specimen preparation, the osmium‐DMSO‐osmium method, which is effective for revealing intracellular structures, has come to be widely used. Techniques for observing smaller objects, such as bacteriophages, viruses, and biological macromolecules, have also been devised in recent years. As a result of these preparation techniques and the availability of the ultra‐high resolution SEM, the application of SEM in biology is expanding rapidly. In this paper, an outline of the ultra‐high resolution SEM, techniques for specimen preparation, findings of some biological materials by these techniques, and guidelines to making the specimens

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00777.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Rat liver microsomes were subfractionated by isopycnic centrifugation in sucrose gradient. The subfractions were assayed for translocation and proteolytic processing of nascent polypeptides in a rabbit reticulocyte lysate programmed with total RNA from human term placenta. The distribution of the translocation and processing of prelactogen through the gradient correlated with that of the microsomal RNA (ribosomes). Microsomes became inactive upon incubation with elastase, but the proteolyzed membranes recovered their activity by recombination with the soluble and active fragment of the docking protein (SRP‐receptor) from dog pancreas. When this fragment was combined with the gradient subfractions, or with the subfractions inactivated by incubation with elastase, the density profile of the translocation activity remained similar to that of RNA. Thus, its distribution cannot be accounted for merely by that of the docking protein; another membrane constituent, still unidentified, is both necessary for translocation of polypeptides and restricted to the rough portions of the endosplamic reticulum. Signal peptidase was assayed in the absence of protein synthesis, by use of preformed prelactogen and detergent‐disrupted microsomes. Its density distribution was also similar to that of RNA. Several components of the endosplamic reticulum now appear to be segregated within restricted areas on either side of the membrane, and to make up a biochemically distinct domain. We propose to call it the ribosomal domain in consideration of its contribution to protein biosynthesis by bound ribosomes. This domain probably accounts for a greater part of the membrane area at the cytoplasmic than at the luminal surface, as postulated earlier to explain how enzymes of the cytoplasmic surface are relatively less abundant in the rough microsomes than those of the luminal surface [Amar‐Costesec A.&Beaufay H. (1981)J. Theor. Biol.89, 217

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00778.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture neurons were shown to express a high degree of tubulin heterogeneity (8 α and 10 β isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 α and 4 β isoforms.After incubation of neuronal and glial cells with3H‐acetate in the presence of cycloheximide, a major posttranslational label was found associated with α‐tubulin and a minor one with β‐tubulin. The acetate‐labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 α and 7 β isoforms, while those of astroglia were resolved into only 2 α and 2 β isoforms. The same α isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated forms(s) of α‐tubulin [38]. Whether acetate‐labeling of α‐tubulin in these cells corresponds to the acetylation of Lys40, as reported forChlamydomonas reinhardtii[30, 33, 34], is discussed according to very recent data obtained by protein sequence analysis.Tubulin phosphorylation was analyzed by incubation of cell cultures with32PO4. No phophorylation of α‐tubulin isoforms was detected. A single β‐tubulin isoform (β′2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mou

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00779.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Monoclonal antibodies raised against the 34‐kD nucleolar protein, B‐36, from the slime moldPhysarum polycephalumhave been used to examine the electron microscopic localization of B‐36 during the cell cycle inPhysarum. During interphase, B‐36 is found primarily in regions corresponding to the dense fibrillar component. This is similar to what has been observed for the putative mammalian homologue of B‐36, fibrillarin. During mitosis, B‐36 remains associated with perichromosomal nucleolar remnants. With the Gautier DNA‐specific staining procedure, the same nucleolar remnants are shown to contain short DNA segments, presumably rDNA molecules. These findings suggest that inPhysarum, where the nucleolus is composed of several hundred extrachromosomal rDNA molecules, the dense fibrillar component and the “NOR” equivalents do not separate during mitosis as in mammalian cells. In addition, the B‐36‐enriched nucleolar remnants appear to be recycled from one c

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00780.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

A central feature of oogenesis in the copepod crustacean,Acanthocyclops vernalis, is the development of a very large nucleolus in the oocytes. This nucleolus appears to be the only source of rRNA for the oocyte, as no helper cells are present. Previous work has suggested that ribosomal DNA sequences other than those found at the morphological nucleolar organizers are participating in the elaboration of this nucleolus. It has been hypothesized that chromatin diminution, which occurs during early embryonic development, may involve the loss of these rDNA sequences, which are needed only for the production of ribosomes during oogenesis. The present study examines the development of the large oocyte nucleolus at the electron microscopic level. Nucleologenesis inA. vernaliswas found to proceed through 5 stages. During the first 3 stages nucleolar morphology resembled that described in other organisms. In the last 2, however, nucleolar morphology changed radically and the nucleolus was seen to increase greatly in size while breaking up into multiple subunits. The subunits initially resemble active nucleoli, although in the last stage, synthesis appears to stop, as the nucleolus was found to consist only of dense areas containing ribosome‐like particles. These observations are consistent with the hypothesis that diminuted DNA contains ribosomal RNA gene

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00781.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Nuclear ribonucleoprotein particles were studied in the central nervous system of the rat after fixation by perfusion with hypotonic‐detergent formaldehyde. Beads‐on‐a‐string structures (polyparticles or chains) formed of 14‐nm granules related by a 7‐nm thick filament were found. These polyparticles are positively contrasted by a preferential method for ribonucleoproteins and they are sensitive to RNase. Perichromatin granules are loosened by this fixation and their internal structure is clearly depicted. They are composed of a tangled mass of 3‐nm thick filaments, but lack 14‐nm granules. The internal filaments of the perichromatin granules display frequent continuities with polyparticles. These results show that polyparticles correspond to perichromatin fibrils visualized in nuclei by standard fixa

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00782.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

The cell cycle phase that mediates the induction of intestinal sucrase‐isomaltase (SI) expression by glucocorticoids was investigated by measuring migration rates of3H‐DNA‐labeled and of SI‐containing epithelial cells by autoradiography and indirect immunofluorescent staining after simultaneous administration of [3H]thymidine and cortisone to 12‐d‐old rat pups. By 24 and 48 h, lead3H‐DNA‐labeled cells had migrated 7.8 and 12.4 cell positions higher on the villus than lead cells expressing SI. Cell migration rates from 12 to 24 h and 24 to 48 h were 0.68 and 0.97 cell position/h. Thus, commitment to SI expression occurred in cells 11.5–12.8 h after the S phase, which is calculated to be in the G1 phase.To determine whether committed cells need to replicate to express SI, cell differentiation was examined in primary cultures of crypt cells originating from corticosterone‐treated rats. About two‐thirds of cultured cells were retarded in the S phase after plating, as judged by no increase of DNA labeling indices, no change in epithelial cell number, and the absence of mitosis (<0.01%). The proportion of cells expressing SI increased from 0 to 6–8% between 12 and 24 h, and reached 48% 48 h after plating on collagen‐coated dishes. SI expression did not occur in cells plated on glass or plastic surfaces. Pulse labeling with [35SH]methionine confirmed thatde novosynthesis of SI occurred in cell cultures. Thus, additional cell cycling of committed cells occuringin vivois not obligatory

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00783.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

A monoclonal antibody against intestinal mucins (5H7) was obtained and used to immunolabel thin frozen sections and Epon‐embedded sections of rabbit jejunum. It recognized a mucin oligosaccharide, the synthesis of which increased during goblet cell migration along the crypt‐villus axis. During the earliest steps in their differentiation, the goblet cells located at the bottom of crypts synthesized mucins devoid of the 5H7 epitope, thus generating unlabeled granules. These unlabeled granules were gradually replaced by more and more labeled granules during the cell maturation. During goblet cell migration along the middle half of the villi, the mucus granules were found to be totally renewed twice. However, some newly formed labeled granules were observed to reach the apical pole of the cells before older unlabeled ones and might have had a faster turnover.At least one glycoconjugate of the goblet cell microvillar membrane also bore the 5H7 epitope. It was rapidly carried from the Golgi apparatus to the apical plasma membrane domain by a transport process that was independent of baseline mucin secret

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00784.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

HT 29‐D4 is a clonal cell line, derived from the human colon adenocarcinoma cell line HT 29, which can be induced to differentiate into enterocyte‐like cells by replacing glucose with galactose in the culture medium (Fantiniet al.[1986],J. Cell Sci.83, 235–249). Both undifferentiated and differentiated HT 29‐D4 cells have been successfully grown to confluency in Costar Transwell permeable chambers. Only HT 29‐D4 cells grown in glucose‐free, galactose‐containing medium were able to form leakproof monolayers, as demonstrated by their ability to prevent diffusion of serum proteins. These monolayers consist of highly polarized epithelial‐like cells with a well organized apical brush border.Transepithelial electrical parameters have been measured under sterile conditions for both types of monolayer. Only HT 29‐D4 monolayers cultured in glucose‐free, galactose‐containing medium were electrically active, with a transepithelial resistance R = 172 ± 46 Ω·cm2, a potential difference PD = 0.35 ± 0.05 mV, apical negative and a short‐circuit current Isc = 2.0 ± 0.4 μA·cm−2. Apical addition of amphotericin B induced a rapid and considerable increase in Isa and PD, which was abolished by basal ouabain.In contrast, HT 29‐D4 cells grown in glucose‐containing medium did not generate any potential difference (PD = 0 mV) and their resistance was very low (R = 34.1 ± 0.9 Ω·cm2).It is concluded from these studies that HT 29‐D4 cells grown in glucose‐free, galactose‐containing medium acquire functional characteristics of epithelia, compared to HT 29‐D4 cells grown in glucose‐containing medium. This unique clonal model system would be of a great interest in studies focused on the mehanisms involved in the establishme

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00785.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

The distribution of the extracellular matrix (ECM) protein, fibronectin (FN), has been examined ultrastructurally in noninjured and injured rat corneal endotheliumin vivoandin vitroby immunoperoxidase cytochemistry. In noninjured endothelia, FN was observed within the rough endoplasmic reticulum (RER) cisternae but not along the cell‐Descemet's membrane (DM) interface. Twenty‐four and 48 h after a circular freeze injury, immunoperoxidase reaction product was detected at the cell‐DM interface as well as within cytoplasmic vesicles and intercellular spaces. By 1 and 2 wk post‐injury, a line of reaction product could still be demonstrated at the cell‐DM interface and evidence for newly deposited basement membrane material was observed in this region. In order to understand whether fibronectin deposition during wound repair was dependent on cytoskeletal influences, organ culture experiments were performed in which the media was supplemented with either 10−8M colchicine or 2.5 × 10−3M cytochalasin B. Without inhibitors, injured corneas cultured for 24 h had FN deposition at the cell‐DM interface similar to thein vivoresults. Corneas cultured in the presence of cytochalasin B also showed FN deposition at the cell‐DM interface.However, when injured endothelia were cultured in the presence of colchicine, no reaction product was observed at the cell‐DM interface, although it could be detected intracellularly within RER. Incubating the tissues in the presence of puromycin abolished all extracellular and intracellular staining. These results indicate that during wound repair, corneal endothelial cells produce fibronectin and deposit it upon Descemet's membrane by a mechanism that may be medi

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00786.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY