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PubMed1983

Fine structure and cytochemistry of lysosomes in the Ito cells of the rat liver.

Ultracytochemical studies of the performic acid-phosphotungstic acid (PFP) reaction and acid phosphatase (ACPase) activity in the Ito cells (fat-storing cells) of the rat liver revealed two kinds of lipid droplets: one surrounded by a structure giving PFP- and ACPase-positive reactions, recognized as a lysosome, the other without such a reactive structure displaying a limiting membrane. To elucidate the function of the lysosomes surrounding lipid droplets, experiments were carried out on the following groups of animals: (1) Vitamin A-deficient rats were fed a normal diet containing vitamin A, and (2) hypervitaminosis A was experimentally induced in previously untreated rats. Lipid droplets were studied in both groups. No lipid droplets reappearing in an early stage after restoration of the regular diet were either membrane-bounded or surrounded by lysosomes. Lipid droplets surrounded by lysosomes could be seen in rats fully restored from vitamin-A deficiency and more frequently in animals suffering from hypervitaminosis A. It seems likely that as a result of the lysosomal activity in the immediate vicinity of the lipid droplets a degradation of the vitamin A-containing lipid droplets takes place in the Ito cells. Therefore, the lysosome-surrounded lipid droplets can be regarded as a sort of autophagolysosome; these lysosomes may play a role in preventing an unrestricted increase in the number and volume of lipid droplets.

Acid Phosphatase↗
PubMedDate not supplied

Diminished axonal transport of glycoproteins in the senescent rat brain.

At various time intervals (10, 15, 20, 25, 30 min) after injection of 3H-fucose into the medial septal nucleus of young adult (3 months old) and senescent (25 months old) Fischer-344 rats, the specific activities of trichloroacetic acid-phosphotungstic acid (TCA-PTA) soluble and insoluble fractions were determined in the medial area of the septum and in three successive rostro-caudal sections of the hippocampal formation containing mainly the dentate gyrus, but also its hilus with fields CA4 and CA3c of the hippocampus. The rate of 3H-fucose incorporation into glycoproteins of the septum did not differ in young adult and senescent rats. Part of the TCA-PTA soluble and insoluble radioactive material was transported through the septo-hippocampal pathway to the dentate gyrus. This transport was inhibited by the injection of colchicine into the septum prior to 3H-fucose injection and was completely blocked by electrolytic lesion of the medial septal nucleus. The arrival time and the amount of the TCA-PTA soluble radioactive material transported to the dentate gyrus did not differ in young adult and senescent rats. However, the TCA-PTA insoluble labelled glycoprotein was transported to the dentate gyrus in a significantly smaller amount and during a longer period of time in the senescent animals. This age-related change may reflect a reduction in amount and/or in rate of axonal transport of glycoproteins in the septo-hippocampal pathway of senescent rats.

Aging↗
PubMed1982

Correlation between growth rate and cytochemistry in Morris hepatomas.

The correlation between the cytochemistry (glycoprotein, glycogen, glucose-6-phosphatase, catalase, alkaline phosphatase) and the growth rate of the fast-growing Morris hepatoma 3924A and the slow-growing Morris hepatoma 9618A was studied by utracytochemical techniques. By the chromic acid-phosphotungstic acid technique, acid glycoprotein is stained in glycocalyx, Golgi saccules and vesicles, and secretory granules of the tumor cells of both hepatomas. However, the hepatoma 3924A cells contain thicker glycocalyx and more numerous glycoprotein-rich granules than hepatoma 9618A cells. Abundant alpha and beta glycogen particles are found in hepatoma 3924A. Moderate glucose-6-phosphatase activity is observed in the cisternae of endoplasmic reticulum and nuclear envelope of hepatoma 9618A, but it is totally absent in hepatoma 3924A. High catalase activity is present in numerous peroxisomes of hepatoma 9618A. Hepatoma 3924A contains only a few catalase-positive microperoxisomes. Weak to moderate alkaline phosphatase is present in the plasma membrane and nuclear envelope of hepatoma 9618A cells, while hepatoma 3924A shows no activity of the enzyme. All the cytochemical parameters except glycoprotein show an inverse relationship with the growth rate of the hepatomas. The higher intracellular glycoprotein content of hepatoma 3924A may be related to differences in cell coat secretion (composition and activity) from the slower-growing hepatoma 9618A

Alkaline Phosphatase↗
PubMed1987

Immunoultrastructural studies of human NK cells: II. Effector-target cell binding and phagocytosis.

The binding of NK cells to a target cell appears to be a necessary step for NK cell-mediated cytolysis. In this report, we demonstrated effector-target binding by immunoelectron microscopy by using monoclonal antibodies against NK cells (Leu-7, Leu-11a) and T-cell subsets (Leu-2a/T8, Leu-3a/T4). The surfaces of NK and K562 cells were characterized by antitransferrin receptor antibody and various lectins. In addition, the controversial phagocytic activity of NK cells was studied by incubation of peripheral blood mononuclear cells with opsonized Staphylococcus aureus and labeling with anti-Leu-7 or anti-Leu-11a antibody. Results showed that only Leu-11a+ cells displayed a broad cell-to-cell contact with the target by a shallow intercellular interdigitation of cytoplasmic projections, while Leu-7+, Leu-2a+, or Leu3a+ cells showed only a partial contact with target without interdigitation. The Leu-11a+ cells were frequently observed in small clusters and in close association with monocytes. Cluster formation and association with monocytes were not observed in other NK and T-cell immunophenotypes. In Leu-11a+ cells conjugated with target cells, membrane-bound granules, small vesicles, parallel tubular arrays, Golgi apparatus, endoplasmic reticulum, and small vacuoles were evident and concentrated toward the target. The surface of NK cells was intensely stained for glycoprotein by chromic acid-phosphotungstic acid, whereas target cells were not stained. Transferrin receptors were stained only on the surface of target cells. Only the lectins RCA and UEA labeled the surfaces of both NK and target cells. Phagocytic vacuoles containing cell debris or fragments and ingested bacteria were found in the cytoplasm of Leu-11a+ cells but not in Leu-7+ cells. NK cells were also found within the cytoplasm of K562 target cells. All these findings suggest that Leu-11a+ cells are the true functional NK cells involved in NK cell-mediated cytolysis, phagocytosis, and emperipolesis. Therefore, the NK cell is probably "a phagocyte in lymphocyte's clothing." The presence of peroxidase in the small vesicles of NK cells and endocytotic vesicles of target cells at the effector-target contact area indicates that cytolytic enzymes or factors derived from NK cells may be transported into the target by endocytosis.

Antibodies, Monoclonal↗
PubMed1977

Mucosubstances in the normal human oesophageal epithelium. A comparison of periodic acid-silver and phophotungstic acid techniques.

Normal human oesophageal epithelium was investigated with the periodic-acid-silver methenamine technique and its variations to demonstrate neutral mucosubstances at the ultrastruct level. The results were compared with the acid phosphotungstic acid method. Neutral mucosubstances were shown in the cell coat and membrane coating granules by both techniques. The silver methods also demonstrated glycogen, the Golgi apparatus and dense bodies. The periodic acid-silver methenamine technique outlined positive material in the intercellular space of the prickle cell layer, but the other silver methods did not.

Epithelium↗
PubMed1979

Methods of heavy metal electron microscopic histochemistry applied to frog lung surfactant.

Four heavy metal staining methods have been applied to frog lung surfactant. Among them, the iodoplatinate method is the only one that almost exclusively visualizes the phospholipid moiety being produced in the lamellated bodies of the pulmonary epithelial cells and forming the backbone of organized structures within the extracellular lining layer. The other three techniques-ruthenium red-osmium tetroxide, osmium tetroxide-ferrocyanide, acidic phosphotungstic acid in chromatic (Rambourg technique)--more or less give electron contrast to glycoproteins and to a lesser extent to the hydrophilic parts of phospholipids. They all show the extracellular lining layer to be a two component system: the content of the lamellar bodies form--when released--membranous configurations, similar to those observed in mammalian lungs; they unfold in an amorphous hypophase, which is apparently secreted by goblet cells of the pulmonary epithelium.

Animals↗
PubMed2004

Ultrastructural characterisation of the host-pathogen interface in white blister-infected Arabidopsis leaves.

In this study transmission electron microscopy (TEM) was used to examine details of the host-pathogen interface in Arabidopsis thaliana cotyledons infected by Albugo candida, causal agent of white blister. After successful entry through stomatal pores, the pathogen developed a substomatal vesicle and subsequently produced intercellular hyphae. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were spherical and 4.5 microm in diameter. Each haustorium was connected to intercellular hyphae by a narrow, slender haustorium neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. No obvious response was observed in host cells following formation of haustoria. Most of the mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cells suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotrophic Oomycete differs considerably from responses to other pathogens such as necrotrophs. Modification of the host plasma membrane (PM) along the cell wall and around the haustoria, was detected by applying the periodic acid-chromic acid-phosphotungstic acid (PACP) staining technique. After staining with PACP, the host PM was found to be intensely electron dense where it was adjacent to the host cell wall and the distal region of the haustorial neck. By contrast, the extrahaustorial membrane, where the host PM surrounded the haustorium, was consistently very lightly stained.

Arabidopsis↗
PubMed1982

An electron microscopic histochemical and X-ray microprobe study of spherites in a mussel.

Transmission electron microscopic, histochemical and X-ray analytical microprobe techniques were used to study the inorganic-organic relationship in the spherites (calcospherules) from the mantle, i.e. subadjacent to the outer mantle epithelium, of the fresh-water mussel Amblema. These structures were shown to contain calcium which could be chelated by the flotation of sections on solutions of either formic acid or ethylene glycol bis-(beta-amino ethyl ether)-N, N1-tetra-acetic acid (EGTA), Analysis of both non-chelated and sections revealed a significant sulfur peak. Chelated spherites were also intensely stained with acid phosphotungstic acid (PTA), Such data is indicative of the presence of an organic glycoprotein (proteoglycan) matrix which could serve to bind mineral ions, thus forming organic-inorganic aggregates for calcium transport and homeostasis. In this regard, the spherites are analogous to both calcium phosphate containing mitochondrial granules and the initial calcification sites in vertebrate mineralizing tissues.

Animals↗
PubMed1995

Effect of essential fatty acid deficiency on G-proteins, cAMP-dependent protein kinase activity and mucin secretion in the rat submandibular salivary glands.

Studies were conducted to determine whether beta-adrenergic cell signalling is altered in submandibular salivary glands (SMSG) is essential fatty acid (EFA) deficiency. Three groups of rats were fed diets which were deficient in EFA (EFAD), marginally deficient in EFA (MEFAD) or contained sufficient amount of EFA (Control). Rats were killed after 20 wk on diets, SMSG were dissected out and cyclic AMP-dependent protein kinase (PKA) activity was measured. The specific enzyme activities were higher in the homogenates and supernatant fractions of the gland from EFAD and MEFAD rats compared with the controls. The relative levels of guanine nucleotide-binding regulatory proteins (Gs and Gi) were also measured in the SMSG membranes of rats fed the 3 diets. The levels of Gs were significantly higher in the EFAD and MEFAD groups than in the controls. No significant differences were observed in the secretion of trichloroacetic acid-phosphotungstic acid (TCA-PTA) precipitable glycoproteins from the SMSG slices among the 3 dietary groups.

Adenylyl Cyclases↗
PubMed1979

Functional characteristics of dispersed rat submandibular cells.

Rat submandibular gland cells have been obtained through enzymatic dispersion using chromatographically purified collagenase (EC 3.4.24.3) and hyaluronidase (EC 3.2.1.35) and gentle mechanical force. The recovery of viable cells after the isolation procedure was 59% on the basis of total glandular DNA content. Approximately 60% of the total cell population consisted of acinar cells; less than 8% were immature granular duct cells; and the remainder were intercalated duct, striated duct, and myoepithelial cells. Most of the acinar cells were in acinar-intercalated duct complexes. The integrity of the isolated cells was substantiated by their exclusion of trypan blue, intracellular electrolyte composition, incorporation of [14C]glucosamine into trichloroacetic acid + phosphotungstic acid precipitable material at a linear rate for 1.5 hr, secretory responses to parasympathomimetic and sympathomimetic stimulation, and morphologic integrity as determined by light and electron microscopy. The cholinergic receptors were characterized through investigation of the net transmembrane flux of K+ in response to carbamoylcholine. The alpha-adrenergic receptors were characterized by investigating the net transmembrane flux of K+ in response to norepinephrine stimulation and the beta-adrenergic receptors were characterized by determining the rate of secretion of 14C-labeled mucin after isoproterenol stimulation. A high degree of sensitivity to both cholinergic and adrenergic secretagogues was observed.

Animals↗
PubMed1976

Association of latent cellulase activity with plasma membranes from kidney bean abscission zones.

Membranes isolated from abscission zones of Phaseolus vulgaris L., cv. Red Kidney, contained cellulase activity. This particulate activity was enhanced 10- to 20-fold by treatment with Triton X-100. Sucrose density gradient analyses of cell fractions showed that the membranes with which cellulase was associated had a peak equilibrium density of 1.16 to 1.17 g/cm(3) which coincided with that of ion-activated ATPase, a marker for plasma membranes. The membrane fraction having the highest cellulase activity also contained a high proportion of plasma membranes as shown by electron microscopy of sucrose density gradient fractions after staining by periodic acid-chromic acid-phosphotungstic acid. It was concluded that the particulate cellulase was associated with the plasma membrane.

Journal Article↗
PubMed1984

Transport of 125I-EGF into milk and effect of sialoadenectomy on milk EGF in mice.

Epidermal growth factor (EGF) (urogastrone) is found in high concentrations in mouse and human milk. The origin of milk EGF is unknown. Milk samples were collected from lactating mice 2-6 h after the intravenous administration of a tracer dose of 125I-labeled EGF. The milk contained relatively high levels of radioactivity of which 35-46% was precipitated by trichloroacetic acid-phosphotungstic acid (TCA-PTA) and 26% by a specific antimouse EGF antiserum. Part of the radioactivity in milk was eluted from a Bio-Gel P-10 column at the point at which pure standard 125I-EGF was eluted. These data indicate that the mammary gland of the lactating mouse is capable of sequestering and transporting 125I-EGF into milk. Administration of a thousand-fold excess of unlabeled EGF caused no reduction in TCA-PTA-precipitable radioactivity in milk samples of mice given 125I-EGF. When mice were given 10 micrograms of unlabeled EGF and milk was collected 4 h later, compared with controls the EGF level in milk was doubled. Administration of EGF had no effect on lactose and protein concentrations in the milk, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no qualitative or quantitative changes in the major milk proteins. Milk collected from lactating mice that were sialoadenectomized 6 mo earlier contained about 50% less EGF compared with controls. After the administration of 125I-EGF high concentrations of radioactivity were also found in the mammary and submandibular glands and in the stomach. In the latter organs, however, 95-96% of the radioactivity was in the acid-soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Potent stimulation of glycoprotein secretion in canine trachea by substance P.

The effects have been investigated of the regulatory peptides, substance P (SP) and bombesin, on the secretion of [14C]glucosamine-labeled trichloroacetic acid-phosphotungstic acid precipitable glycoproteins by canine tracheal explants. SP (10(10) to 10(-7) M) induced a dose-dependent increase in secretion of high-molecular-weight (greater than 2 X 10(6) radiolabeled glycoproteins predominantly from the submucosal glands. On a molar basis, SP [median effective concentration (EC50) = 8.2 X 10(-10) M] was about 1,000-fold more potent than methacholine (EC50 = 6.3 X 10(-7) M). Bombesin (10(-10) to 10(-4) M) had no effect on glycoprotein secretion. The time course of SP effect was characterized by an initial stimulation of glycoprotein secretion followed by a period of inhibition, suggesting that it rapidly exhausts a pool of glycoprotein, possibly that present within the duct lumen of the submucosal gland. Consistent with this are the findings that SP-induced secretion of glycoprotein was augmented by preincubation with methacholine while methacholine-induced secretion was diminished by preincubation with SP. Our findings show that SP is a potent stimulant of airway glycoprotein secretion in vitro and suggest that it acts by increasing the rate of clearance of mucus from the ducts of the submucosal gland, possibly by induced constriction of the secretory tubules and collecting duct. A role is discussed for SP in mucus hypersecretion induced by local axonal reflexes in the airway mucosa.

Animals↗
PubMed1981

An electron microscopic histochemical and analytical X-ray microprobe study of calcification in Bruch's membrane from human eyes.

Transmission electron microscopy, electron microprobe analysis, high temperature microincineration, and electron microscopic histochemical procedures were used to study the electron-dense deposits characteristic of the macular aspect of aged human eyes. These inorganic deposits were rich in calcium and phosphorus and selectively removed by flotation on formic acid. The amorphous decalcified masses showed a significant sulfur peak and were readily stained with acidic phosphotungstic acid. The latter observations are indicative of the presence of organic matrical proteoglycan. Such data may be a further indication that proteoglycans are retained at sites of calcification.

Calcinosis↗
PubMed1982

Cartilage calcification: an ultrastructural, histochemical, and analytical x-ray microprobe study of the zone of calcification in the normal avian epiphyseal growth plate.

Sections from the zone of calcification of ruthenium red-fixed normal avian epiphyseal growth plates were analyzed by various morphological (histochemical) and analytical techniques. Calcium and phosphorus were identified in the chondrocyte pericellular rim, the uncalcified extracellular (territorial) matrix, and in both the peripheral and central aspects of the calcified accumulations within extracellular matrix. Cartilage proteoglycan, as determined by ruthenium binding, positive staining with acidic phosphotungstic acid, and the X-ray spectroscopic detection of sulfur, was identified in the same four zones. Thus, it appears that proteoglycans, in some form, are indeed retained at sites of biological calcification. Additionally, these macromolecules, synthesized in chondrocytes, may be involved in extracellular calcium translocation.

Animals↗
PubMed1985

A new method for scanning and transmission electron microscopy of synaptic vesicles isolated from the cerebral cortex.

Spheroid and flattened synaptic vesicles were isolated from the brain homogenate of guinea pigs by a modified purification method. For scanning and transmission electron microscopy, a simple dipping method of preparation was developed and used. The purest and richest fraction of synaptic vesicles was obtained from a 0.1 M sucrose fraction of density gradients. The pellets of synaptic vesicles were easily resuspended without aggregate after ultracentrifugation at 40,000 rpm for 5 min. The isolated synaptic vesicles were dispersed as a monolayer on the surface of a copper grid covered with Formvar membrane. Adequate contrast was obtained by metal impregnation of specimens and gold coating at magnifications as high as 100,000 times using an acceleration voltage of 25 to 40 kV. The specimens were fixed in 0.75% glutaraldehyde (0.1 M phosphate buffer, pH 7.3) and then postfixed in 1% osmium tetroxide. After dipping for 1 to 2 min each in tannic acid, phosphotungstic acid, lead citrate and uranyl acetate, they were dehydrated with graded ethanol and coated with gold by ion sputtering at 400 to 560 volts for 4 min. The preparation method is reported on and technical problems are discussed.

Animals↗
PubMed1989

Acetylcholine in neurons and paraneurons: a histochemical study.

Several years ago we proposed a method for the localization of acetylcholine in the cholinergic nerve terminals. The method is based on a rapid precipitation of quarternary ammonium cations (such as acetylcholine or choline) by molybdic or tungstic heteropolyanions (such as phosphomolybdic acid, phosphotungstic acid and silicotungstic acid). The insoluble salts formed can be directly visualized under the electron microscope. In the synaptic vesicles, acetylcholine was localized as point-like precipitates, while the membranes were well preserved. Since these fixations are based on a rapid ionic interaction, the term "ionic fixation" was proposed. The ionic fixation performed on motor end-plates in various physiological conditions, provided different forms of cytochemical precipitates of acetylcholine (point-like, spot-like, diffuse or laminar). In contrast with the numerous physiological and neurochemical investigations into the acetylcholine mechanism of neurons, very little is known about paraneurons. The historical evolution of paraneuron research, more or less bound to that of the sympathetic paraganglion and of the APUD cells may explain this lack of results. In certain physiological conditions, acetylcholine can be revealed in paraneurons and seems to reflect a plasticity of the cells, as already observed in sympathetic ganglion cells. This paper further overviews the cholinergic mechanisms of exocrine secretory cells. The presence of a high cholinesterase activity exist in most of these exocrine cells, as far as we could verify. In addition to cholinesterase activity, the presence of acetylcholine, probably related to exocrine cell metabolism, was investigated with our histochemical method.

Acetylcholine↗