Gene/Protein Disease Symptom Drug Enzyme Compound
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The application of scanning electron microscopy to the study of cell surfaces is limited in intact tissues, because extracellular material may often obscure the details of nonluminal surfaces. To remove connective tissue elements we have treated human skin and both kidney, and an autonomic ganglion of the rat with hydrochloric acid and collagenase. Regional variations in the basal surface of the nephron are noted following removal of the basement membrane. The basilar interdigitations of the cells of the proximal tubule appeared as parallel ridges encircling the tubule. Ridges on the parietal epithelium of Bowman's capsule were randomly arranged and alternated with smooth surfaces. The dermal surface of the human epidermis has an alveolar or honeycomb appearance due to the elevation of the epidermal ridges and numerous pits for the dermal pegs. At higher magnifications the basal surface of cells of the stratum germinativum possessed numerous and irregular projections. Neurons with their processes are evident in the autonomic ganglion. The soma of the neurons are enclosed by flattened satellite cells. Irregular spaces between opposed satellite cells are interpreted as regions for the passage of processes related to the ganglion cells. Nodes of Ranvier were clearly seen along nerve fibers. Some pitting of the nerve fibers was also noted. The HCl-collagenase method has the advantage of the removal of collagen and basement membrane while preserving the structural integrity of the cell surface.
Anat Rec 1976 Aug
PMID:Scanning electron microscopy of cell surfaces following removal of extracellular material. 18 20

Electron microscopic observations and measurements were made on thin-sectioned chromatin fibers and fibrils obtained from nuclei of mature chicken erythrocytes. The nuclei were isolated in low ionic strength gum arabic and octanol then extracted sequentially with (1) 0.14 M NaCl, (2) 0.25 N HCl, (3) buffer saturated phenol, (4) hot 5% SDS and 0.14 M 2-mercaptoethanol and, (5) 0.4 N NaOH. The amount of nuclear protein removed at each of the first four extraction steps was 1, 86, 3 and 11% of the total, respectively. Each extract was characterized by electrophoretic profiles. At each extraction the chromatin was fixed by adding large quantities of a mixture of equal volumes of sodium cacodylate buffered 8% (w/v) glutaraldehyde (pH 6.8) and 2% OsO4 (w/v), directly into (1) an aliquot of the chromatin in extraction fluid, and (2) an aliquot of the chromatin after water washing and swelling. Three size classes of chromatin structure were seen in thin sections prepared for high resolution transmission electron microscopy and stained with uranyl acetate and lead citrate. A thick fiber of about 25 + nm diameter was the predominant large fiber seen in freshly isolated nuclei or in nuclei after salt extraction. This 25 + nm fiber has a substructure consisting of 3.2-5.2 nm diameter fibrils. After water swelling of such freshly isolated or salt extracted nuclei a fiber of about 10 nm diameter was the predominant large fiber instead of the 25 nm diameter fiber. The HCl extraction step which is known to remove histones, caused the disappearance of both the 25 nm and the 10 nm fibers. High magnification (600,000 x) micrographs of the chromatin at all procedural steps, except the last NaOH step, reveal the fibril to be omnipresent. This fibril tends to decrease somewhat in diameter during the protein extraction steps to a 2.5 nm diameter fibril after the hot SDS extraction. A fibril of 2.5 nm diameter is expected of naked double helical DNA stained with a positive stain. The NaOH, which is known to denature DNA, completely destroyed the remaining fibril. We inerpret our results to indicate that the larger chromatin fiber seen in micrographs of thin-sectioned chromatin has a fibrillar substructure which probably represents a double coil of native DNA which may have a thin protein coating of its own. The latter fibril may in turn be wrapped around a hydrophobic histone domain, perhaps reflected in the 10 nm diameter fiber which is seen upon swelling of the chromatin. This 10 nm diameter fiber is thought to be further packaged by folding into the 25 + nm diameter chromatin fiber most frequently reported in thin sections of eukaryotic cell nuclei in situ.
Anat Rec 1979 Aug
PMID:Chromatin substructure: an electron microscopic study of thin-sectioned chromatin subjected to sequential protein extraction and water swelling procedures. 47 16

Changes in ultrastructure and cytoskeletal organization by avian oxyntic cells, at the onset of HCl secretion, were analysed. Cells in resting state, induced by fasting and cimetidine, were compared with histamine stimulated secreting cells. Ultrastructural studies were done by transmission electron microscopy; the distribution of prekeratin, myosin, and filamin-like protein, by immunofluorescence; and that of F-actin using FITC-phalloidin. Resting cells show short pericellular clefts. These are increasingly deepened in secreting cells by a reorganization of the lateral cell borders involving displacement of the junctional complexes toward the cell base and incorporation of the tubular system to the luminal plasma membrane. In secreting cells, the processes of the secretory surface are concentrated in a pericellular groove. Histamine stimulation induces a drastic redistribution of cytoskeletal proteins. In chicken oxyntic cells, in addition to the F-actin cytoskeleton associated with the membranes of the secretory surface, there is a cytoskeletal ring containing F-actin, myosin, and a filamin-like protein, located at the level of the junctional complexes. In resting cells, filaments and masses of cytoskeletal matrix are associated with the zonula adherens. In secreting cells, the junctional complexes maintain their association with the filamentous ring, while the amorphous matrix is replaced by microfilaments that support the processes of the luminal surface. Intermediate filaments form a peripheral ring probably associated with the zonula adherens, and project from the ring toward the cell cytoplasm. Thus, with the onset of HCl secretion, the apical cytoskeletal ring of resting cells displaces toward the cell base. A role for this cytoskeletal ring in the changes in shape parallel to HCl secretion is discussed.
Anat Rec 1990 Oct
PMID:Redistribution of membranes and cytoskeletal proteins in chicken oxyntic cells during the HCl secretory cycle: ultrastructural and immunofluorescence study. 170 Jun 49

Muscle spindles in the tenuissimus muscle of mature golden Syrian hamsters were examined by conventional and high-resolution scanning electron microscopy (HRSEM). For conventional SEM, entire muscles were first fixed in 2.5% buffered glutaraldehyde. Spindles were then isolated with a dissecting microscope under darkfield illumination and postfixed in 1.0% OsO4. Some spindles were treated with 8 N HCl at 60 degrees C to clearly expose intrafusal fiber surfaces once the outer capsular sheath was mechanically disrupted. Preparation for HRSEM included aldehyde/osmium fixation and freeze-cleavage in liquid N2. The cytosol and certain cellular elements were also selectively extracted by immersion in 0.1% OsO4 for varying time intervals. In these preparations, the capsular sleeve showed a multilayered pattern of vesicle-laden cells with variant surface topography in different regions, including filopodia and small bristle-like surface-projections. An interlacing three-dimensional network of collagen fibrils intervened between the capsular lamellae. Within the spindles, sensory and fusimotor nerve endings closely adhered to the outer surfaces of intrafusal fibers. Sensory nerve terminals were enveloped by a prominent external lamina, and those that were cleaved open contained a plethora of elongated mitochondria that ran parallel with the longitudinal axis, along with vesicles, axoplasmic filaments, and lysosomes. Multiple adhesion sites between the sensory nerve membrane and the underlying sarcolemma of the intrafusal fiber were also observed in select regions. Fusimotor nerve endings were covered externally by processes of Schwann cells and their axoplasm was filled with a multitude of cellular organelles and synaptic vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Anat Rec 1991 Jun
PMID:Muscle spindle ultrastructure revealed by conventional and high-resolution scanning electron microscopy. 186 95

When they were turned out to grass in May 1987 for their first season, 10 calves were dosed with a 5 x 750 mg oxfendazole pulse release bolus (OPRB) and a monensin sodium rumen delivery device (RDD); eight calves received one OPRB; 10 calves received one RDD and eight calves received neither bolus. Each group was set-stocked on individual paddocks which had been grazed during the previous season by cattle which developed clinical parasitic gastroenteritis and bronchitis (husk). In July, before they were due to be moved to new pastures in mid-summer, and before they were dosed strategically with levamisole HCl, some of the calves not dosed with an OPRB succumbed to clinical parasitic gastroenteritis and husk and received emergency anthelmintic treatment, after which no further clinical episodes occurred. The 'dose and move' strategy was implemented in early August after which both groups not dosed with an OPRB were set-stocked together until the trial ended on October 14, 147 days after turn out. The two groups of calves which had received the OPRB were also moved to new pasture and set-stocked together until the end of the trial. No evidence of clinical helminthiases developed in either of the two groups of calves dosed with OPRBs and their faecal worm egg and larval counts, and plasma pepsinogen activities remained low. They gained significantly more weight than the two groups of calves not dosed with OPRBs (P less than 0.001). The bolus types were compatible and induced no untoward side-effects when used together.
Vet Rec 1989 Jul 15
PMID:Concurrent use of the oxfendazole pulse release bolus and the monensin rumen delivery device in young grazing cattle. 277 30

Ultrastructure and three-dimensional architecture of the papillary layer and associated capillaries in the enamel organ of 2-3-month-old kittens were examined by means of routine thin sections, freeze-fracture, and scanning electron microscopy of the tissues digested by HCl-collagenase and of vascular corrosion casts. Outwardly, the papillary layer formed gently sloping upheavals, but did not show prominent papillary ridges. The papillary cells were characterized by a high concentration of intramembranous particles on the plasma membrane P-face, by numerous hemi-annular gap junctions between the cell process of one papillary cell and the cell body of another host cell, and by annular gap junctional vesicles in the subsurface cytoplasm. Some annular gap junctions appeared partially degenerated. These findings led us to speculate that these annular gap junctions are produced by the endocytosis of gap junctional membranes from the cell surface into the subsurface cytoplasm. Capillaries were distributed on the enamel organ within shallow furrows between the papillary upheavals. A part of these capillaries penetrated deeply into the enamel organ but did not contact the ameloblasts. The endothelial walls of the capillaries were pierced with many endothelial fenestrations, especially when facing the papillary layer. The endothelial cell also contained numerous micropinocytotic vesicles throughout its entire cytoplasm. These findings suggest that the papillary cells and associated capillaries are highly specialized for transport of solutes and molecules between the vascular region and the enamel organ during the phase of enamel maturation.
Anat Rec 1986 Apr
PMID:An ultrastructural study of the papillary layer and its vascular bed in the kitten enamel organ. 351 41

We have purified the beta subunit of the DNA polymerase III holoenzyme to homogeneity from an overproducing strain (Blanar, M., Sandler, S., Armengod, M., Ream, L., and Clark, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4622-4626). From this procedure we can obtain 100 mg quantities of protein. The beta isolated from the overproducer is indistinguishable from that isolated from wild-type cells in terms of its activity and molecular weight. Partial amino acid sequence analysis has confirmed the DNA sequence of the dnaN gene (Ohmori, H., Kimura, M., Nagata, T., and Sakakibara, Y. (1984) Gene (Amst.) 28, 159-170) and established the sites for initiation and termination of translation. No processing that removes amino acid residues from beta occurs since the active protein begins with the initiating methionine and terminates at the position predicted from the DNA sequence. Our knowledge of the precise amino acid composition has been used to determine the extinction coefficient of beta to be 17,900 and 18,700 cm-1 M-1 at 280 and 277 nm, respectively. The extinction coefficient at 280 nm is reduced to 14,700 cm-1 M-1 under denaturing conditions in guanidine HCl. Conditions have been optimized so that 1 N-ethylmaleimide residue can be incorporated per beta monomer with full preservation of activity.
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PMID:Chemical characterization and purification of the beta subunit of the DNA polymerase III holoenzyme from an overproducing strain. 352 43

Enamel contains two categories of biochemically characterized proteins. Amelogenins are dissociated from enamel without physical disruption of the tissue whereas enamelins are obtained only when the crystallites are dissolved. Ultrastructural visualization of these proteins was attempted using routine electron microscopy and freeze-fracture replicas. Fresh, fixed, and 4.0 M guanidine-HCl-extracted samples of enamel from the secretory (young) and maturation (maturing) stages were compared. Decalcified and stained thin sections of fixed enamel revealed intercrystallite particulate material and "crystallite ghosts" which were identical to the crystallites themselves in young enamel and which corresponded to the periphery of the crystallites in maturing enamel. In contrast, 4.0 M guanidine-extracted enamel contained no intercrystallite particulate material but only "crystallite ghosts." Globular particles observed in freeze-fracture replicas of fresh and fixed enamel samples were also removed by 4.0 M guanidine extraction. Incubation of guanidine-extracted enamel with albumin and ovalbumin solutions restored the globular particles. It was concluded that amelogenins are the nonstructural, heterodispersed particulate material in the intercrystallite space. Enamelins constitute the integral template protein which initially provides for elongation of enamel crystallites. They then regulate the continuous growth in width and thickness during maturation and are progressively displaced to the periphery. The illusion that these "protein ghosts" are contained within the crystallite profile can be explained by the parallelepiped shape of the crystallite segment in thin sections.
Anat Rec 1985 May
PMID:Morphological studies on the distribution of enamel matrix proteins using routine electron microscopy and freeze-fracture replicas in the rat incisor. 407 36

At the muscle-tendon junction of skeletal muscle fibers the structural interface between muscle cell and connective tissue is amplified by tapering, by indentation, and by surface folding. The precise form taken by the surface folds has been unknown due to a lack of studies on the three-dimensional geometry of the muscle-tendon junction. Analysis of this region by scanning electron microscopy, using conventional preparative techniques, is uninformative because the muscle surface is covered by connective tissue. Removal of the connective tissue from individual murine muscle fibers by incubation of fixed fibers in hot HCl, followed in some instances by treatment with collagenase, permits SEM analysis of the uncovered fiber ends. The muscle fiber end is characterized by surface specializations in the form of anastamotic cylindrical folds. Transmission electron micrographs of cross sections and of serial longitudinal sections of muscle fiber ends confirm that the SEM observations are correct.
Anat Rec 1985 Sep
PMID:Three-dimensional structure of the murine muscle-tendon junction. 407 57

Gastric K+-NPPase represents a partial reaction of the (K+-H+)ATPase system, which is considered to be the proton pump in mammalian parietal cells. In the present paper, K+-NPPase activity was cytochemically studied by the method of Mayahara et al. (1980) in gastric glands of birds, amphibia, and mammals, either in the resting state induced by cimetidine or after stimulation of HCl secretion by histamine. The gastric K+-NPPase cytochemical reaction was localized only in oxyntic cells of the gastric mucosa in the three species tested. The subcellular distribution of the K+-NPPase reaction product drastically changes with the secretory state of HCl. In resting cells, the K+-NPPase staining is associated with the membranes of the endocellular tubular system while in HCl-secreting cells, it is associated with the plasma membrane of the elaborate secretory surface characteristic of this functional state. The above results demonstrate that the same enzymatic activity, which is associated with the gastric proton pump, is present in both membranous systems of the oxyntic cell secretory pole. This fact supports the proposal that the tubular system represents a membrane reserve that inserts the proton pump into the luminal plasma membrane in vertebrate oxyntic cells under the action of HCl secretagogues.
Anat Rec 1984 Dec
PMID:Redistribution of gastric K+-NPPase in vertebrate oxyntic cells in relation to hydrochloric acid secretion: a cytochemical study. 609 93


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