scholarly journals PRESERVATION OF Na-K-ACTIVATED AND Mg-ACTIVATED ADENOSINE TRIPHOSPHATASE ACTIVITIES OF AVIAN SALT GLAND AND TELEOST GILL WITH FORMALDEHYDE AS FIXATIVE

1970 ◽  
Vol 18 (4) ◽  
pp. 251-263 ◽  
Author(s):  
STEPHEN A. ERNST ◽  
CHARLES W. PHILPOTT
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Electron Microscopic Examination ◽  
Salt Gland ◽  
Chloride Cells ◽  
Secretory Cells ◽  
Salt Glands ◽  
Electron Microscopic ◽  
Fixed Tissue ◽  
Gill Filaments

The effect of glutaraldehyde and formaldehyde fixation on the level of biochemically demonstrable Na-K-adenosine triphosphatase (Na-K-ATPase) and Mg-ATPase of avian salt glands and teleost gill filaments was studied. Sections, 100-200 µ, prepared with the Smith-Farquhar tissue chopper, were fixed for varying periods, homogenized and assayed for ATPase activity. Fixation of salt gland tissue with 0.5% glutaraldehyde for 40-60 min completely inhibited the Na-K-ATPase activity and reduced the level of Mg-ATPase by 85%. In contrast, fixation with 2 or 3% formaldehyde, prepared from paraformaldehyde, for 60-90 min resulted in a loss of only 30% of the Na-K-ATPase activity and 65% of the Mg-ATPase activity. Similar results were obtained with gill filaments. In addition, Na-K-ATPase of formaldehyde-fixed tissue retained an obligatory requirement for Na+ and K+ and was fully sensitive to ouabain. Electron microscopic examination of formaldehyde-fixed tissue, sectioned with either the tissue chopper or in the cryostat and incubated in the Wachstein-Meisel medium, showed excellent morphologic preservation. Reaction product deposition (presumably due to Mg-ATPase) was associated with the extracellular side of the plasma membrane in the secretory cells of the salt gland and over the mitochondrial matrix of chloride cells present in the gill epithelium.

Changes in Na+/K+-ATPase expression during adaptive cell differentiation in avian nasal salt gland

1997 ◽  
Vol 200 (13) ◽  
pp. 1895-1904 ◽  
Author(s):  
JP Hildebrandt
Keyword(s):  
Salt Stress ◽  
Cyclic Amp ◽  
Atpase Activity ◽  
Time Course ◽  
Atp Hydrolysis ◽  
Salt Gland ◽  
Secretory Cells ◽  
Salt Glands ◽  
Atpase Subunit

Chronic salt stress in ducklings (Anas platyrhynchos) resulted in a sustained accumulation of cyclic AMP in the secretory cells of the nasal salt glands. Adaptive increases in the activity of the Na+/K+-ATPase, measured as ATP hydrolysis rates in freshly isolated tissue, were observed after 12 h of salt stress. This change in enzyme activity was associated with increases in protein abundance in the - as well as in the ss-subunit of Na+/K+-ATPase and an increase in ss-subunit glycosylation. We investigated whether the increase in the cytosolic cyclic AMP concentration and the adaptive changes in Na+/K+-ATPase activity were causally related. Using an organotypic tissue culture system for salt gland slices from unstressed (naive) ducklings, we produced similar changes in Na+/K+-ATPase activity and subunit abundance by treating cultured tissue with drugs that elevate cytosolic cyclic AMP levels (forskolin, 8-CPT-cAMP) during a 15 h culture period. Protein synthesis assays using cultured tissue revealed that elevations in cytosolic cyclic AMP level mediate increases in Na+/K+-ATPase subunit abundance by slowing down the degradation of ATPase subunits. This increase in the amount of enzyme protein was associated with a significant increase in Na+/K+-ATPase activity in tissue homogenates. The time course of these changes in cyclic-AMP-treated cultured tissue resembled that observed in salt-stressed intact animals, indicating that the elevation in cyclic AMP level in salt gland tissue may constitute a portion of the signalling events ultimately leading to the adaptive increase in Na+/K+-ATPase activity in vivo.

TRANSPORT ADENOSINE TRIPHOSPHATASE CYTOCHEMISTRY II. CYTOCHEMICAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE ACTIVITY IN THE SECRETORY EPITHELIUM OF THE AVIAN SALT GLAND

1972 ◽  
Vol 20 (1) ◽  
pp. 23-38 ◽  
Author(s):  
STEPHEN A. ERNST
Keyword(s):  
Phosphatase Activity ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Salt Gland ◽  
Ultrastructural Localization ◽  
Defined Medium ◽  
Secretory Cells ◽  
Cytochemical Localization ◽  
Fixed Tissue ◽  
Nitrophenyl Phosphate

A cytochemical procedure is described for the ultrastructural localization of K-dependent, ouabain-sensitive nitrophenyl phosphatase activity in avian salt gland. Cryostat sections (50 µ) of paraformaldehyde-fixed tissue were incubated in a kinetically defined medium containing: 5 mM p-nitrophenyl phosphate, 10 mM MgCl2, 10 mM KCl, 100 mM Tris-HCl buffer (pH 8.5 or 9.0) and 20 mM SrCl2 to precipitate hydrolyzed phosphate. After incubation at room temperature, the sections were treated with Pb(NO3)2 to convert SrPi to PbPi precipitates for visualization in the electron microscope. Reaction product was localized on the cytoplasmic side of the secretory cell lateral and basal plasma membranes. Little, if any, reaction product was associated with the apical surfaces of the secretory cells or with endothelial surfaces of capillaries. Appropriate control experiments indicated that deposition of reaction product was dependent on Mg and K and was sensitive to ouabain. Furthermore, nonenzymatic hydrolysis of nitrophenyl phosphate did not occur in the medium, and deposition of artifactually produced precipitates did not resemble deposition of enzymatically produced precipitates. The relationship of this localization to transport adenosine triphosphatase cytochemistry is discussed, and the physiologic implications of the localization for tracing the route of active Na transport in the salt gland are considered.

A New Human Breast Tumor Cell Line

1975 ◽  
Vol 33 ◽  
pp. 388-389
Author(s):  
R.E. Nordquist ◽  
R.M. Wasik ◽  
P.J. Riggs ◽  
P.L. Munson ◽  
F.B. Schafer
Keyword(s):  
Electron Microscopy ◽  
Cell Line ◽  
Calf Serum ◽  
Tumor Cell Line ◽  
Electron Microscopic Examination ◽  
Epithelial Cell Line ◽  
Electron Microscopic ◽  
Fixed Tissue ◽  
Excised Tissue ◽  
Caucasian Female

An infiltrating ductal cell carcinoma was removed from the breast of a postmenopausal Caucasian female. The excised tissue was divided into three parts; one part for electron microscopy, one part for tissue culture and the remainder frozen for immunological studies.The tissue for culture was minced finely with sterile razor blades and cultured in Falcon flasks containing Eagel's MEM supplemented with 10% heat denatured fetal calf serum. The tissue for electron microscopy was fixed in 6.25% glutaraldehyde in 0.1 M PO4 buffer plus 5% sucrose and postfixed in 1% OsO4 in the same buffer. The fixed tissue was dehydrated in graded ethanol and embedded in Spurr.The tissue which was cultured began to grow out after approximately six weeks and became a continuous epithelial cell line which was designated BOT-2 (Breast Original Tumor). Electron microscopic examination revealed that these cells had epithelial characteristics, i.e. the presence of tonofilaments and well formed desmosomes.

Biogenesis of Plasma Membranes in Salt Glands of Salt-Stressed Domestic Ducklings: Localization of Acyltransferase Activity

1972 ◽  
Vol 11 (3) ◽  
pp. 855-873
Author(s):  
A. M. LEVINE ◽  
JOAN A. HIGGINS ◽  
R. J. BARRNETT
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Salt Gland ◽  
Secretory Cells ◽  
Salt Glands ◽  
Acyltransferase Activity ◽  
Structural Localization ◽  
Differentiated Cells ◽  
Golgi Membranes

In response to salt water stress there is a marked increase in the plasma membranes of the epithelial secretory cells of the salt glands of domestic ducklings. In the present study, the fine-structural localization of the acyltransferases involved in synthesis of phospholipids has been investigated in this tissue during this increased biogenesis of plasma membranes. The specific activity of the acyltransferases of the salt gland rose in response to salt stress, and this preceded the rapid increase in weight and cellular differentiation. After the weight increase of the gland became established, the specific activity of the acyltransferases declined, but the total activity remained constant. Salt gland tissue fixed in a mixture of glutaraldehyde and formaldehyde retained 35% of the acyltransferase activity of unfixed tissue. Cytochemical studies of the localization of acyltransferase activity in fixed and unfixed salt gland showed reaction product associated only with the lamellar membranes of the Golgi complex. This localization occurred in partially differentiated cells from salt-stressed glands to the greatest extent; and to only a small extent in cells of control tissue from unstressed salt glands. Omission of substrates resulted in absence of reaction product in association with the Golgi membranes. In addition, vesicles having limiting membranes morphologically similar to the plasma membrane occurred between the Golgi region and the plasma membrane in the partially differentiated cells. The phospholipid component of the plasma membrane appears therefore to be synthesized in association with the Golgi membranes and the membrane packaged at this site from which it moves in the form of vesicles to fuse with the pre-existing plasma membrane.

scholarly journals Studies in vitro on the effects of 1H,2H,4H(5H)-octafluorocyclohexane and 1H,4H(2H)-nonafluorocyclohexane on enzymes and organelles

1969 ◽  
Vol 114 (4) ◽  
pp. 785-792 ◽  
Author(s):  
Jayasree Nath ◽  
H G Bray
Keyword(s):  
Adenosine Triphosphatase ◽  
Marked Decrease ◽  
Reductase Activity ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Toxic Compound ◽  
Marked Inhibition ◽  
Liver Homogenates ◽  
Nadh Cytochrome

A comparison has been made of the effect of 1H,2H,4H(5H)-octafluorocyclohexane, which is highly toxic (LD50 17mg./kg. in rats), and of 1H,4H(2H)-nonafluorocyclohexane, which is relatively non-toxic (LD50>440mg./kg. in rats), on the respiration of rat liver homogenates and mitochondria in vitro. 1H,2H,4H(5H)-Octafluorocyclohexane strongly inhibited the respiration of both homogenates and mitochondria, but neither compound had any significant effect on glycolysis or on glutamate dehydrogenase or NADH–cytochrome c reductase activity. 1H,2H,4H(5H)-Octafluorocyclohexane, however, caused a very marked inhibition of cytochrome oxidase activity, causing an almost complete lesion in this region of the respiratory chain. 1H,4H(2H)-Nonafluorocyclohexane was without effect in this respect. A marked decrease in turbidity of mitochondrial suspensions at 520nm. was caused by addition of both compounds, the effect being greater with 1H,2H,4H(5H)-octafluorocyclohexane. ATP, Mg2+ and bovine serum albumin did not reverse these changes. Mitochondrial adenosine triphosphatase activity was increased twofold by the toxic compound, but only slightly by the non-toxic compound. Electron-microscopic examination of mitochondria treated with 1H,2H,4H(5H)-octafluorocyclohexane revealed gross morphological damage, whereas the effect of 1H,4H(2H)-nonafluorocyclohexane appeared to be merely to cause swelling. The results obtained account, to some extent at any rate, for the toxic effects of 1H,2H,4H(5H)-octafluorocyclohexane.

Potassium secretion by nasal salt glands of desert lizard Sauromalus obesus

1987 ◽  
Vol 253 (1) ◽  
pp. R83-R90 ◽  
Author(s):  
T. J. Shuttleworth ◽  
J. L. Thompson ◽  
W. H. Dantzler
Keyword(s):  
Atpase Activity ◽  
Intraperitoneal Injection ◽  
Salt Gland ◽  
Salt Glands ◽  
Fourfold Increase ◽  
Gland Weight ◽  
Desert Lizard ◽  
Potassium Secretion ◽  
Sauromalus Obesus

Potassium secretion by the nasal salt glands of the herbivorous desert lizard Sauromalus obesus was determined in vivo by a new technique. Intraperitoneal injection of KCl rapidly increased the potassium secretion rate from 0.28 to 15.35 mumol X 100 g-1 X h-1. A second identical intraperitoneal injection, given 15 h after the first, further increased potassium secretion to 50.09 mumol X 100 g-1 X h-1. This was associated with a doubling of plasma K+ concentration and salt gland Na+-K+-adenosinetriphosphatase (ATPase) activity. Neither salt gland weight or residual (Mg2+) ATPase activity were affected. In an isolated perfused head preparation, potassium secretion from the nasal salt glands was stimulated from 0.99 to 10.76 mumol X 100 g-1 X h-1 by methacholine and to 14.68 mumol X 100 g-1 X h-1 by forskolin. In this perfused preparation, simultaneous determination of salt gland perfusion flow (using radiolabeled microspheres) and the rate of potassium secretion revealed that the secreting glands removed 68% of the perfusing potassium ions. Calculations indicated that secretion at the maximal rate observed in vivo would necessitate a fourfold increase in the rate of blood flow to the gland.

Juvenile Spindle Cell Carcinoma

1979 ◽  
Vol 87 (5) ◽  
pp. 573-577 ◽  
Author(s):  
William P. Potsic ◽  
R. Beverly Raney ◽  
Billy E. Buck ◽  
Steven W. Fischer
Keyword(s):  
Cell Carcinoma ◽  
Microscopic Examination ◽  
Spindle Cell ◽  
Treatment Plan ◽  
Electron Microscopic Examination ◽  
Spindle Cell Carcinoma ◽  
Electron Microscopic ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Formalin Fixed

Spindle cell carcinoma is an uncommon variant of squamous cell carcinoma that is rarely seen in children. The spindle cell pattern is frequently confused with sarcomas. A case of spindle carcinoma in a 14-year-old boy is presented. He is the youngest patient, to the authors' knowledge, with spindle cell carcinoma of the maxilla. Electron microscopic examination is helpful to define the epithelial nature of the spindle cells and can be performed on formalin-fixed tissue. Electron microscopic examination is essential to formulate an optimal treatment plan.

scholarly journals LEAD ION AND PHOSPHATASE HISTOCHEMISTRY II. EFFECT OF ADENOSINE TRIPHOSPHATE HYDROLYSIS BY LEAD ION ON THE HISTOCHEMICAL LOCALIZATION OF ADENOSINE TRIPHOSPHATASE ACTIVITY

1966 ◽  
Vol 14 (10) ◽  
pp. 702-710 ◽  
Author(s):  
HAROLD L. MOSES ◽  
ALAN S. ROSENTHAL ◽  
DAVID L. BEAVER ◽  
SHIRLEY S. SCHUFFMAN
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Lead Nitrate ◽  
Histochemical Localization ◽  
Lead Ion ◽  
Membrane Localization ◽  
Fixed Tissue ◽  
Plasma Membrane Localization ◽  
Hydrolysis Of

The lead method of Wachstein and Meisel for the histochemical localization of adenosine triphosphatase (ATPase) involves the incubation of sections of fixed tissue in reaction mixtures containing ATP, lead nitrate, magnesium sulfate and a Tris-maleate buffer, pH 7.2. Both fixation and the presence of lead ion were shown to inhibit tissue ATPase activity markedly and to inactivate the sodium- plus potassium-dependent membrane ATPase. In addition, recent studies have demonstrated that lead ion, in the concentration used in the Wachstein-Meisel system, will catalyze the hydrolysis of ATP. Studies on the effect of this nonenzymatic reaction on the histochemical localization of ATPases demonstrated that plasma membrane localization occurred only with lead and ATP concentrations which gave significant nonenzymatic hydrolysis of ATP by lead. In addition, nuclear and mitochondrial localization without accompanying plasma membrane localization could be obtained in formalin-fixed tissue with decreased concentrations of lead or with increased concentrations of ATP in the reaction mixture. The amount of lead-catalyzed hydrolysis was in the same order of magnitude as fixed tissue ATPase activity and could quantitatively account for the amount of phosphate needed to give recognizable localization of lead salt deposits in sections of fixed tissue.

Salt stress increases abundance and glycosylation of CFTR localized at apical surfaces of salt gland secretory cells

1994 ◽  
Vol 267 (4) ◽  
pp. C990-C1001 ◽  
Author(s):  
S. A. Ernst ◽  
K. M. Crawford ◽  
M. A. Post ◽  
J. A. Cohn
Keyword(s):  
Salt Stress ◽  
Single Cells ◽  
Salt Gland ◽  
Cell Protein ◽  
Secretory Cells ◽  
Salt Glands ◽  
Cl Secretion ◽  
Peptide Antibody ◽  
Nacl Secretion ◽  
Apical Membranes

Osmotic stress elicits hypertonic NaCl secretion and promotes structural and biochemical differentiation in avian salt glands. In addition to cholinergic control, Cl- secretion is stimulated by vasoactive intestinal peptide (VIP), suggesting that the cystic fibrosis transmembrane conductance regulator (CFTR) may be present and that its expression may be regulated by chronic salt stress. Anion efflux, assayed by 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence changes in single cells, was stimulated by VIP or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. Immunoblots with a COOH-terminal peptide antibody to human CFTR revealed approximately 170- and approximately 180-kDa bands in lysates from control and salt-stressed glands, respectively. Both variants reduced to approximately 140 kDa after N-glycanase digestion and gave identical tryptic phosphopeptide maps after immunoprecipitation and phosphorylation by protein kinase A. CFTR was localized to apical membranes by immunofluorescence and, additionally, to subapical vesicles by immunoelectron microscopy. Salt stress induced an approximately twofold increase in CFTR abundance/cell protein (approximately 5-fold/cell) and intensified apical membrane immunofluorescence. For comparison, Na+ pump expression increased approximately fourfold per cell protein with little change in actin. Thus differentiation induced by salt stress is accompanied by alteration in CFTR abundance and glycosylation. Upregulation of CFTR likely contributes to increased efficiency of Cl- secretion.

scholarly journals Myosin from pancreatic acinar carcinoma cells. Isolation, characterization and demonstration of heavy- and light-chain phosphorylation

1987 ◽  
Vol 247 (3) ◽  
pp. 513-518 ◽  
Author(s):  
T K Watanabe ◽  
E R Kuczmarski ◽  
J K Reddy
Keyword(s):  
Ionic Strength ◽  
Atpase Activity ◽  
Heavy Chain ◽  
Electron Microscopic Examination ◽  
Acinar Cell Carcinoma ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Light Chains ◽  
Post Translational Modification ◽  
Electron Microscopic

Myosin has been identified in a variety of non-muscle cells, and is believed to play a role in maintenance of cell shape, locomotion, cytokinesis, exocytosis and other cellular functions. In this paper we describe the purification of myosin from a pancreatic acinar-cell carcinoma of the rat which forms solid tumours, but retains many differentiated functions. The purified myosin was composed of a 200,000 Da heavy chain and two or three classes of light chains. Electron-microscopic examination of rotary-shadowed preparations revealed that individual molecules had two globular heads and a long tail measuring approx. 149 nm. The myosin was soluble in high-salt buffers and became sedimentable as the ionic strength was lowered. Examination of negative-stained preparations showed that this sedimentable myosin consisted of short, bipolar, thick filaments which had a strong tendency to aggregate in a head-to-head manner. The ATPase activity of the purified myosin was stimulated by EDTA or Ca2+, but not by Mg2+. In low ionic strength the Mg2+-dependent ATPase activity was activated by muscle f-actin. The pancreatic myosin bound to actin and could be dissociated by the addition of MgATP. Myosin purified from cells cultured in media containing [32P]Pi was phosphorylated on one of the light chains as well as the heavy chain. Thus pancreatic acinar cells contain a typical non-muscle myosin, and the subunits of this molecule are subject to post-translational modification by phosphorylation.

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