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To further characterize Sertoli cell-germ cell junctional specializations seminiferous tubules from sexually mature Sprague-Dawley rats were dissociated by enzymatic and mechanical methods. Ultrastructural analysis of cell suspensions prepared by incubation in collagenase alone or by mechanical methods revealed that spermatids remained attached to Sertoli cells or Sertoli cell fragments. Such cellular associations were found only between Sertoli cell fragments and spematids in which the developing acrosome had made contact with the plasma membrane (step 8 and subsequent steps of spermiogenesis). Furthermore, the fragments were confined to that region of the plasma membrane over the acrosome. The Sertoli cell half of this adhesive site displayed the typical elements of Sertoli cell junctions, filamentous bundles and associated cisterna of endoplasmic reticulum, in apposition to the spermatids. The spermatids demonstrated no surface specializations at the attachment sites. In contrast, in cell suspensions prepared with trypsin, spermatids were free of attachments to Sertoli cells or their fragments. These results demonstrate that: (1) the junctions act to bind cells together, (2) adhesive type contact is established between Sertoli cells and spermatids at step 8 and subsequent steps of spermiogenesis, (3) contact is restricted to the spermatid plasma membrane over the acrosome, and (4) spermatids can be freed from the junctional specializations by treatment with trypsin.
Anat Rec 1979 Jan
PMID:Characterization of Sertoli cell-germ cell junctional specializations in dissociated testicular cells. 21 84

Injury in the periphery of the lung was studied in rats given intratracheal trypsin. Mitotic figures were noted in the alveolar epithelium, and colchicine enhanced the yield. Of 65 mitotic figures examined in detail, 46 were present in the alveolar epithelium. Epithelial mitoses were confined to type II cells, many of which contained lamellar inclusions, abundant rough endoplasmic reticulum, and luminal microvilli. Mitoses were not seen in type I cells or in less well differentiated epithelial cells, but they were noted in occasional endothelial and interstitial cells. Colchicine-arrested type II cells were distinguished by radially disposed chromosomes surrounding pairs of centrioles and randomly oriented microtubules. In addition, many of these cells showed a pronounced cortical zone, devoid of microvilli, and others showed cytotoxic degenerative changes. These observations indicate that the alveolar epithelium is renewed exclusively by division of differentiated type II cells.
Anat Rec 1979 Aug
PMID:Mitoses in peripheral pulmonary epithelium: the effect of colchicine. 47 12

Fragments of adult rabbit lung, composed chiefly of terminal airway obtained by a trypsin digestion technique were maintained on collagen-coated cellulose sponges in Ham's F12 medium. Cell-sponge associations were examined with light microscopy, scanning and transmission electron microscopy over a period from 6 to 28 days. After an initial 24- to 48-hour period of cell migration from the airway fragment, sponge matrices became lined with cells suggestive of alveolar macrophages. After one week in culture, cysts appeared to be composed entirely of type 2 epithelial cells. These were characterized by a microvillous apical border and an elaborate junctional complex. The lumen of these cysts contained both myelin-like lamellar configurations and tubular myelin structures such as have been described from pulmonary washings. Consistent with the age of the sponge cultures, one or more cyst types described as young, middle and late could be found simultaneously. Middle aged cysts showed signs of active secretion into the lumen. Late cysts showed changes in the epithelium comprising the cyst wall suggestive of a cell type intermediate between type 1 and type 2 epithelial cells.
Anat Rec 1977 Jun
PMID:Ultrastructure of in vitro type 2 epithelial cell cysts derived from adult rabbit lung cells. 55 27

A lambda DNA supercoil system has been developed to study the effects of colicin E2 on DNA in vivo. Colicin E2, a protein antibiotic synthesized by strains of coliform bacteria that carry the Col E2 plasmid, had as its most conspicious effect damage to the DNA of sensitive strains. Colicine E2 attacks the supercoiled molecul formed by labeled lambda DNA in superinfected cells as well as it attacks the bacterial DNA. The rate and extent of acid solubilization of the lambda supercoils and of host bacterial DNA induced by E2 treatment are nearly identical. Treatment of superinfected cells with colicin E2 results in the progressive conversion of lambda DNA supercoils to open circles and/or linear full lenght molecules, and subsequently to fragments less than full lambda in size. The first endonucleolytic reactions are single-strand and or double-strand breaks. The rate of supercoil breakdown as well as the final percent supercoils remaining unconverted, the size of the final lambda fragments, and the extent of solubilization are dependent on the multiplicity of colicin used. Additions of trypsin to E2-treated superinfected cells results in a cessation of further breakdown of the lambda molecules, presumably as a result of digestion of accessible colicin molecules. Energy is essential for an early event in colicin E2 action. The host enzymes, endonuclease I and Rec BC, may be instrumental in the nucleolytic process caused by colicin E2: endonuclease I in reaction preceding cell killing and Rec BC in a secondary degradation of the bacterial DNA.
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PMID:The action of colicin E2 on supercoiled lambda DNA. I. Experiments in vivo. 114 58

The surface coat of syncytial trophoblast from term human placentas was studied using cytochemical methods (colloidal iron, alcian blue-lanthanum nitrate, dialyzed iron) in coordination with tissue enzyme digestions (trypsin, neuraminidase) and sialic acid analyses. The presence of at least two highly acidic anionic components that contribute significantly to the surface negativity of trophoblast has been demonstrated. The first of these, sialic acid, was removed with neuraminidase. Tissue digestion with this glycosidase was accompanied by a decrease in trophoblast surface staining with colloidal iron, a decrease in tissue sialic acid, and an increase in the concentration of sialic acid in the incubating medium. Results from methylation experiments were consistent with the presence of sialic acid. The second anionic component(s) was identified by removal with trypsin of a glycocalyx constituent that stained with both colloidal iron and lanthanum. After trypsinization, tissue sialic acid levels were not significantly different from control values, and no detectable sialic acid was present in the incubating medium. The identity of this anionic component has not been established. Both sialic acid and nonsialic acid acidic components are distributed in higher density on membrane of microvilli than on intermicrovillous surface membrane. In addition, the sialic acid moieties appear to be clustered in the glycocalyx.
Anat Rec 1976 Feb
PMID:The nonuniform distribution of acidic components on the human placental syncytial trophoblast surface membrane: a cytochemical and analytical study. 124 83

Three-dimensional alteration of fibrillar matrix in the rat mandibular condylar cartilage was investigated with a high-resolution scanning electron microscope (SEM) and it was determined whether alterations correlate with developing occlusion and advancing age. Two important SEM techniques of DMSO freeze-cracking and treatment with trypsin and hyaluronidase were employed to remove interfibrillar proteoglycans and disclose fibril arrangement. Our SEM investigation demonstrated that collagen fibrils in the fibrous zone covering hyaline-cartilaginous area in the condyle are thicker (50 to 80 nm in diameter) than the fibrils (30 to 50 nm in diameter) that predominantly constituted an interterritorial fibrillar matrix (IFM) in the area. While the thick fibrils had a distinct striation of about 55 nm periodicity, the thin fibrils had no distinguishable striation. The thick fibrils having a periodic striation of about 60 nm was found along with the thin fibrils, also in the IFM in the aged rats and in the deep IFM, but were considerably less than the thin fibrils. The fibrils in the fibrous zone and IFM were disorderly arranged at 19-day-insemination age. In 1-week-old rats whose incisors erupted, the fibrils constituting the fibrous zone altered from disordered to ordered arrangement. The IFM in these rats took the form of a network. Incorporation of small fibrillar bundles into the fibrillar network was seen in 2-week-old rats whose upper and lower first molars erupted. In 8-week-old rats whose molars had erupted completely, the IFM completely occupied by regularly oriented fibrils appeared additionally.(ABSTRACT TRUNCATED AT 250 WORDS)
Anat Rec 1992 Dec
PMID:Ultrastructural alteration of cartilaginous fibril arrangement in the rat mandibular condyle as revealed by high-resolution scanning electron microscopy. 145 52

Cauda epididymal guinea pig spermatozoa are arranged in rouleaux, with the sperm heads stacked one on top of the other; the plasma membranes over the apical segment of the acrosomes of adjacent sperm are linked and form non-fusigenic "junctional" zones. A complex structural and temporal sequence of membrane fusions occurs during the acrosome reaction of guinea pig sperm in rouleaux. In this study, we have devised a procedure for dispersing the rouleaux and isolating a population of single, motile guinea pig sperm, and have investigated the ultrastructural features of the acrosome reaction in single sperm to determine if the pattern of membrane fusions is different from sperm in rouleaux. The rouleaux were dispersed using trypsin, and damaged cells were removed by passing the sperm suspension through a glass bead column; a population of 70-90% motile, acrosome-intact, single sperm was obtained. Sperm were then induced to undergo lysolecithin-mediated, "synchronous" acrosome reactions, and processed for transmission electron microscopy. The acrosome reaction involved a complex sequence of membrane fusions between the plasma membrane (PM) and outer acrosomal membrane (OAM). On the convex surface of the apical segment, sheets of hybrid membrane and parallel arrays of hybrid membrane tubules formed; filaments were associated with the luminal surface of the residual OAM in these regions. Hybrid membrane vesicles were produced on the concave surface of the apical segment, but fusion was delayed relative to the convex surface. In the principal segment, branching arrays of hybrid membrane tubules formed and later vesiculated.(ABSTRACT TRUNCATED AT 250 WORDS)
Anat Rec 1991 Feb
PMID:Ultrastructural analysis of the acrosome reaction in a population of single guinea pig sperm. 201 5

The capacity of the dental pulp to form calcified tissue was examined in papilla cells dissociated from first molar tooth germs of the neonatal mouse and isografted in the spleen for up to 7 days. To obtain papilla cell populations without odontoblasts, pulpal mesenchyme was isolated mechanically from the enamel organ after 0.1% trypsin treatment and rolled on a membrane filter. On day 3 after transplantation, the grafted papilla cells had changed into large, spindle-shaped cells, and initial calcification with needle-like crystals began in association with the collagenous matrix surrounding those cells. On day 7 after transplantation, the spindle cells transformed into odontoblast-like cells containing well-developed secretory organelles, and irregular, but nontubular, calcified tissues were commonly observed surrounding the extracellular collagenous matrix. The calcified tissue matrix with cellular inclusions displayed a structure similar to that of osteodentin. During this period, an intense positive reaction for alkaline phosphatase (ALPase) activity was demonstrated along the cell membranes of the odontoblast-like cells aligned at the periphery of forming calcified tissue. Enzymatic activity could not be detected on the cells incorporated completely into osteodentin-like matrix. The present results show that the papilla cell population transplanted into the spleen formed osteodentin-like material, thus demonstrating the capacity of papilla cells to produce calcified tissue.
Anat Rec 1990 Mar
PMID:Calcification capacity of dental papilla mesenchymal cells transplanted in the isogenic mouse spleen. 232

Previous work has demonstrated that adult newt cardiac myocytes possess a proliferative ability in response to an experimentally induced injury, in vivo. This study describes an in vitro model in which the proliferative events of the adult cardiac myocyte may be studied. Ventricles were minced and then enzymatically dissociated in a Ca++- and MG++-free salt solution containing 0.5% trypsin and 625 U/ml of CLS II collagenase for 8 to 10 hours at 25 degrees C. Enzyme digests were preplated and then cultured on bovine corneal endothelial-derived basement membrane "carpets" in either serum-free or serum-supplemented modified Leibovitz's medium for up to 30 days. Light and transmission electron microscopic characterization demonstrated that a majority of the myocytes underwent an initial period of disorganization characterized by a "rounding up" of the cell and a loss of myofibrillar organization. Once the myocytes had attached to the culture substratum they began to spread out, underwent a reassembly of their contractile elements, resumed spontaneous contractions, and demonstrated ultrastructural evidence of protein synthesis. Mitosis was observed in several myocytes 8 to 15 days following isolation. In 15-day serum-supplemented and serum-free cultures, 6.5% +/- 0.9% and 8.1% +/- 1.4% of the myocytes were binucleated, respectively. These results demonstrate that adult newt ventricular myocytes can be successfully placed into primary culture and are capable of undergoing mitosis. This work may be considered as a foundation for future investigations which will focus on the mechanisms which control cardiac myocyte proliferation.
Anat Rec 1989 May
PMID:Primary cell culture and morphological characterization of ventricular myocytes from the adult newt, Notophthalmus viridescens. 265 85

We have investigated the possibility that the complex patterns of fluorescence associated with spermatids of the ground squirrel labeled with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin) are due to the presence of filamentous actin within the spermatids themselves rather than to actin in attached Sertoli cell ectoplasmic specializations, as previously reported (J. Cell Biol., 100:814-825). Enzymatic treatments (trypsin, DNAase 1) freed Sertoli cell ectoplasmic specializations from spermatids and resulted in a loss, from the spermatids, of the complex fluorescence patterns, suggesting that the latter were generated by labeled actin in ectoplasmic specializations. Moreover, ectoplasmic specializations that were detached enzymatically from spermatids demonstrated the same fluorescence patterns as those emitted from spermatids in the intact or mechanically fragmented seminiferous epithelium. Most spermatids, however, do display a weak and diffuse pattern of fluorescence that changes during spermatogenesis and that is localized between the acrosomal cap and nucleus. S-1 decoration confirmed this subacrosomal localization and further demonstrated that the actin in adjacent Sertoli cell ectoplasmic specializations is arranged in a unipolar fashion. We conclude that the complex patterns of actin fluorescence associated with mechanically isolated spermatids are a superimposition of both Sertoli cell and germ cell actin; however, the latter is either poorly detected or not detected at all when Sertoli cell ectoplasmic specializations overlie the germ cells.
Anat Rec 1986 Aug
PMID:Distribution of actin in Sertoli cell ectoplasmic specializations and associated spermatids in the ground squirrel testis. 352 80


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