Freeze-fracture replication of organized tissue without cryoprotection. (17/34186)

Fresh pieces of rat liver and pancreas were rapidly frozen without prior chemical fixation or cryoprotection, and replicated folloing freeze-fracture. Replicas revealed small peripheral areas free of ice crystals or damage and, within such areas, general ultrastructural morphology was essentially similar to that seen in conventionally processed material. On fracture faces of plasma and nuclear membranes a population of less prominent particles in addition to conventional membrane-associated particles was seen, and smooth areas devoid of particles of any type were seen on some nuclear membranes. These smooth areas did not appear to be similar to smooth areas allegedly arising as artifacts of conventional processing. Tight junctions and gap junctions appeared as they do in cryoprotected specimens. The results provide a base-line for assessing the possible effects of processing steps or agents on the ultrastructure of organized tissues as revealed in freeze-fracture replicas.  (+info)

The postnatal development of the alimentary canal in the opossum. I. Oesophagus. (18/34186)

The oesophageal epithelium of the newborn opossum generally is two to three cells in depth and in some regions appears pseudostratified. By the 9th postnatal day the epithelium shows two distinct strata. Ciliated cells and occasional goblet cells also are observed within the epithelium during this stage and in subsequent stages. Cilia persist in the oesophagus of the adult opossum, but are restricted to the depths of the transverse folds found in the distal part of the organ. The epithelium covering the transverse folds of the adult likewise has an immature appearance. By 4-5 cm (ca. 20 days), the epithelium has assumed a more mature appearance and is of greater depth. This and later stages show three basic strata: a germinal layer, a spinous layer and, adjacent to the lumen, a flattened layer of cells that retain their nuclei. The epithelium throughout the postnatal period and in the adult does not undergo complete keratinization. The oesophageal glands begin as outgrowths from the epithelium just prior to 4-5 cm (ca. 20 days). The glands continue their development throughout the remainder of the postnatal period. The secretory units of the oesophageal glands of the the major portion of the secretory elements, and a light, rounded cell type which is less numerous and which occupies the terminal portions of the secretory units. Secretory material of the former appears complex, consisting of both neutral and acid glycoproteins. The secretory product of the light cell type is unknown at present. Both cell types are encompassed by myoepithelial cells. The relationship of the mitotic sequences to the observations made by microscopic examination of the developing oesophagus is discussed.  (+info)

Localization of sodium bicarbonate cotransporter (NBC) protein and messenger ribonucleic acid in rat epididymis. (19/34186)

An acidic environment is important for sperm maturation in the epididymis and also helps to maintain mature sperm in an immotile state during storage in this organ. Both an Na+/H+ exchanger and an H+ATPase have been implicated in this process. The H+ATPase is concentrated in specialized apical (and/or narrow) and clear cells of the epididymis, while the Na+/H+ exchanger has not yet been localized in situ. As in other proton-secreting epithelia, bicarbonate transport occurs in the epididymis, where it is implicated in luminal acidification. In this study we used an antibody raised against a fusion protein (maltose-binding protein: MBP-NBC-5) from the C-terminus of the recently cloned rat kidney Na+/HCO3- cotransporter (NBC) to localize this protein in the epididymis and vas deferens of the rat. The distribution of the respective mRNA was mapped by in situ hybridization. NBC message was strongly expressed in the initial segment and the intermediate zone of the epididymis, and the NBC-5 antibody gave a strong basolateral staining in both principal cells and apical/narrow cells in this region. Western blotting revealed a single band at about 160 kDa in the epididymis. The intensity of staining as well as mRNA levels decreased in the cauda epididymidis and in the vas deferens, where only weak staining was seen. Basolateral NBC may function in parallel with apical proton secretion to regulate luminal acidification and/or bicarbonate reabsorption in the excurrent duct system.  (+info)

An intact sperm nuclear matrix may be necessary for the mouse paternal genome to participate in embryonic development. (20/34186)

We have been interested in determining the minimally required elements in the sperm head that are necessary in order for the paternal genome to participate in embryogenesis. We used an ionic detergent, mixed alkyltrimethylammonium bromide (ATAB), plus dithiothreitol (DTT) to remove the acrosome and almost all of the perinuclear theca, leaving only the sperm nucleus morphologically intact. We also tested the stability of the sperm nuclear matrix by the ability to form nuclear halos. Sperm nuclei washed in freshly prepared 0.5% ATAB + 2 mM DTT completely decondensed when extracted with salt, but nuclei washed in the same buffer that was 1 wk old, and then extracted with salt, produced nuclear halos, indicating stable nuclear matrices. When we treated sperm heads with freshly prepared ATAB+DTT and injected them into oocytes, none of the oocytes developed into live offspring. In contrast, sperm heads treated in the same way but with 1-wk-old ATAB+DTT solution could support development of about 30% of the oocytes to live offspring. Electron microscopy demonstrated that most of the perinuclear theca had been removed in both cases. These data suggest that at least in the mouse, the only component of the spermatozoa that is crucial for participation in embryologic development is the sperm nucleus with a stable nuclear matrix.  (+info)

Development and cytodifferentiation of the rabbit pars intermedia. II. Neonatal to adult. (21/34186)

Material from pars intermedia obtained from rabbits ranging from the second week post-partum to the adult stage, and including specimens from pregnant animals, was studied. The rate of cell division became greatly reduced early in postnatal) development. The commonest type of cell (the pars intermedia-glandular cell) becomes increasingly PAS-positive during the early stages of development. Although by 35 days differentiation of all the ACT-type cells is complete, the pars intermedia-glandular cells take as long as 53 days to mature. The epithelioid border of the hypophysial cleft persists throughout life, commonly containing dark cells. A ciliary fringe frequently appears in neonates and persists in pregnancy. Possible functions of such cilia are discussed. Throughout development the fine structure of the vasculature was studied. Secretory granules resembling those within the cells were seen in and around the blood vessels, and the mode of endocrine secretion in the pars intermedia tissue is discussed. The pars intermedia-glandular cells of the pregnant rabbits appeared hyperactive. The functional significance of the mammalian pars intermedia is discussed.  (+info)

Langerhans cells in the human oesophagus. (22/34186)

The dendrite cells of Langerhans, first identified in the epidermis, have now been observed in the middle and superficial layers of the normal human oesophageal mucosa. They exhibit typical Langerhans granules, but no desmosomes and tonofilaments. They often have irregular indented nuclei, with a relatively pale cytoplasm contrasting with that of the adjacent squamous cells. These cells are sometimes difficult to distinguish from intra-epithelial lymphocytes, which are also encountered in the oesophageal mucosa and which share certain ultrastructural characteristics with Langerhans cells.  (+info)

Recombinant human peroxisomal targeting signal receptor PEX5. Structural basis for interaction of PEX5 with PEX14. (23/34186)

Import of matrix proteins into peroxisomes requires two targeting signal-specific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structural information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1-78) with a very high affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1-78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these complexes revealed that PEX5 possesses multiple binding sites for PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A pentapeptide motif that is reiterated seven times in PEX5 is proposed as a determinant for the interaction with PEX14.  (+info)

Molecular basis for the enterocyte tropism exhibited by Salmonella typhimurium type 1 fimbriae. (24/34186)

Salmonella typhimurium exhibits a distinct tropism for mouse enterocytes that is linked to their expression of type 1 fimbriae. The distinct binding traits of Salmonella type 1 fimbriae is also reflected in their binding to selected mannosylated proteins and in their ability to promote secondary bacterial aggregation on enterocyte surfaces. The determinant of binding in Salmonella type 1 fimbriae is a 35-kDa structurally distinct fimbrial subunit, FimHS, because inactivation of fimHS abolished binding activity in the resulting mutant without any apparent effect on fimbrial expression. Surprisingly, when expressed in the absence of other fimbrial components and as a translational fusion protein with MalE, FimHS failed to demonstrate any specific binding tropism and bound equally to all cells and mannosylated proteins tested. To determine if the binding specificity of Salmonella type 1 fimbriae was determined by the fimbrial shaft that is intimately associated with FimHS, we replaced the amino-terminal half of FimHS with the corresponding sequence from Escherichia coli FimH (FimHE) that contains the receptor binding domain of FimHE. The resulting hybrid fimbriae bearing FimHES on a Salmonella fimbrial shaft exhibited binding traits that resembled that of Salmonella rather than E. coli fimbriae. Apparently, the quaternary constraints imposed by the fimbrial shaft on the adhesin determine the distinct binding traits of S. typhimurium type 1 fimbriae.  (+info)