Distinct distribution of four Ca2+-dependent subtypes of protein kinase C in rat olfactory bulb; definite expression of betaII-subtype in the accessory olfactory bulb

The localization of four subtypes of Ca2+-dependent protein kinase C (PKC) in the main and accessory olfactory bulb was examined by immunocytochemistry by using specific antibodies against each PKC subtype. In the main olfactory bulb, alpha-PKC was densely localized in a large number of granule cells and in a few tufted cells, and faint immunoreactivity was seen in some periglomerular cells. betaI-PKC was intensely found in periglomerular cells and tufted cells. gamma-PKC immunoreactivity was present in the external plexiform layer, the internal plexiform layer, and the granular layer, but the immunoreactivity was found only in the neuropils. Little, if any, betaII-PKC was seen in the main olfactory bulb. On the other hand, the intense immunoreactivity for betaII-PKC was seen in periglomerular cells of the accessory olfactory bulb. The betaI-PKC and gamma-PKC were also present in periglomerular cells of the accessory olfactory bulb, while alpha-PKC was localized only in granule cells. Double staining study in the accessory olfactory bulb showed that betaII-PKC was present in the GABAergic periglomerular cells, while betaI-PKC localized to the non-GABAergic periglomerular cells; gamma-PKC was expressed in both GABAergic and non-GABAergic cells. These findings suggest that four calcium-dependent subtypes of PKC play different roles in the olfactory bulb and definite expression of betaII-PKC strongly suggested the involvement of this subtype in a specific function in the accessory olfactory bulb.     

Cellular and subcellular localization of kainic acid in the marine red alga Digenea simplex

Polyclonal antibodies specific for the excitatory amino acid, kainic acid (KA), were raised in rabbits. The antibody recognized KA but did not cross-react with other structurally related amino acids, including glutamate. We used this anti-KA antibody to localize KA immunohistochemically in the KA-producing red alga Digenea simplex. KA immunoreactivity was most dense in the fine cylindrical thallus, which covers the middle to upper part of the alga. The cortical cells, but not the inner layers of the main axis, and cells of the rhizoid were also stained with this antibody. The presence of KA in cells that cover the surface of the alga might reflect its role in chemical defense. At the subcellular level, KA immunoreactivity was most intense in the nucleus, pit plugs, and the electron-dense areas denoted as “granule bodies”, which were found only in the pericentral cells of the thallus. This research was supported by Ministry of Education, Culture, Sports, Science and Technology to R.S. (13660206).     

Localization of Allatostatin-Producing Cells in Larval Gut of the Potato Worm Agrius convolvuli

ABSTRACT This study has been carried out to investigate localization of allatostatin-producing cells in the gut of Agrius convolvuli using an immunocytochemical method. Allatostatin producing cells could not be found in the foregut and hindgut, but they were abundantly distributed only in the posterior part of midgut. These endocrine cells had typically columnar shape, with secretory surface positioned to muscular layer of midgut wall and also showed different intensity of immunoreactivity to AST, mainly with strong or moderate intensity.     

Uptake of circulating hyaluronic acid by the rat liver

Summary The present study deals with the localization and development of S-100 protein-like immunoreactivity in the retina, ciliary body and iris of human fetuses. In the retina, numerous astrocytes, densely distributed in the nerve-fiber layer and ganglion-cell layer, were stained strongly with the S-100 antiserum. The first immunoreactive astrocytes occurred at the posterior pole of the retina and spread gradually outward and toward the ora serrata with increasing age. Müller cells were not immunoreactive for S-100 during development, except in the retina of the latest fetus examined. S-100 immunoreactivity was also found in the nonpigmented ciliary epithelium and posterior epithelium of the iris, both of which are developed from the inner wall of the optic cup. On the other hand, the pigmented epithelium extending from retina to iris, derived from the outer layer of the optic cup, was free of S-100 immunoreactivity.     

Distribution and developmental expression of lamin-like immunoreactivity in the central nervous system.

Lamins are the major proteic constituents of the nuclear lamina, the innermost layer of the nuclear membrane. The immunolocalization of lamins in the rat central nervous system was studied using polyclonal antibodies. Besides an ubiquitarious localization in the nuclear membranes of neurons and glial cells, an intense lamin-like immunoreactivity was found in the soma and dendrites of cerebellar Purkinje cells. The same specific reaction was also observed in the human cerebellum. Experiments performed in newborn animals demonstrated that the cytoplasmic expression of lamins in Purkinje cells begins during postnatal development.     

Disruption-free imaging by Raman spectroscopy reveals a chemical sphere with antifouling metabolites around macroalgae

Investigations of the surface chemistry of marine organisms are essential to understand their chemically mediated interactions with fouling organisms. In this context, the concentration of natural products in the immediate vicinity of algal surfaces, as well as their biological activity, are of particular importance. However, due to lack of appropriate methods, the distribution of compounds within the chemical sphere around marine algae is unknown. This study demonstrates the suitability of confocal resonance Raman microspectroscopy for the determination of metabolites around algal surfaces with a micrometer resolution. The spatial distribution of carotenoids in the diffusion boundary layer of the brown alga Fucus vesiculosus and the green alga Ulva sp. was determined using the disruption-free optical method. A gradient of carotenoids was determined within 0 to 150 μm from the surface of the algae, thereby demonstrating the release of the non-polar metabolites involved in antifouling processes. The differences in the carotenoid composition of the brown and green algae were reflected in the spectra. Resonance Raman microspectroscopy also allowed visualization of the lateral distribution of fucoxanthin on the algal surface and localization of concentration maxima within a 50 × 50 μm(2) area. The results from this work show clearly that established dipping techniques suitable for the extraction of the diffusion boundary layer of macroalgae only provide an average of the local strongly variable concentrations of metabolites on algal surfaces.     

D-aspartate oxidation by rat and bovine renal peroxisomes: an electron microscopic cytochemical study

D-amino acid oxidase, a peroxisomal enzyme, and D-aspartate oxidase, a potential peroxisomal enzyme, share biochemical attributes. Both produce hydrogen peroxide in flavin-requiring oxidative reactions. Such similarities suggest that D-aspartate oxidase may also be localized to peroxisomes. Definitive identification of D-aspartate oxidase as a peroxisomal enzyme depends, however, on visualization at the electron microscopic level. Using incubation conditions shown to be specific for the enzyme in biochemical studies, this report extends the cytochemical localization of D-amino acid oxidase to bovine renal peroxisomes, and shows that D-aspartate can be oxidized by rat and bovine renal peroxisomes. An unexpected finding was the sensitivity of both D-amino acid oxidase activity (proline specific) and D-aspartate oxidase activity to inhibition by agents used in biochemical studies to discriminate between the two enzyme activities. Therefore, it is possible that, in the cytochemical system used in this study, (a) either D-proline and D-aspartate are substrates for only one enzyme or (b) the two enzymes have additional overlapping biochemical properties.     

D-Aspartate stimulation of testosterone synthesis in rat Leydig cells

D-Aspartate increases human chorionic gonadotropin-induced testosterone production in purified rat Leydig cells. L-Aspartate, D-,L-glutamate or D-,L-asparagine could not substitute for D-aspartate and this effect was independent of glutamate receptor activation. Testosterone production was enhanced only in cells cultured with D-aspartate for more than 3 h. The increased production of testosterone was well correlated with the amounts of D-aspartate incorporated into the Leydig cells, and L-cysteine sulfinic acid, an inhibitor of D-aspartate uptake, suppressed both testosterone production and intracellular D-aspartate levels. D-Aspartate therefore is presumably taken up into cells to increase steroidogenesis. Intracellular D-aspartate probably acts on cholesterol translocation into the inner mitochondrial membrane, the rate-limiting process in steroidogenesis.     

Localization of human airway trypsin-like protease in the airway: an immunohistochemical study

Human airway trypsin-like protease (HAT) has been isolated from mucoid sputum of patients with chronic airway diseases. In order to clarify the cellular source of this novel protease in the human airway, we examined the localization of immunoreactive HAT in bronchial tissues obtained at surgery and fixed in 4% paraformaldehyde using an extremely sensitive immunohistochemical technique called a catalyzed signal amplification method and a monoclonal antibody against recombinant HAT. HAT immunoreactivity was demonstrated in cytoplasm of ciliated cells of bronchial epithelium and/or at the basal part of cilia. No positive reaction was found in submucosal glands or mast cells. The heterogeneous distribution of HAT immunoreactivity within the bronchial epithelium indicates that its expression might be changeable and that it might be closely related to the physiological status of the airway epithelium. Non-specific but intense reaction caused by endogenous avidin-binding activity (EABA) was selectively detected in submucosal glands, but was effectively blocked by successive treatments with avidin and biotin. These results indicate that HAT may be synthesized in the ciliated cells and that it may play some physiological roles within the epithelial layer and on the airway surface. It is necessary to keep in mind that some cells show strong EABA, especially when a highly sensitive immunohistochemical technique is applied.     

干出和沉水不同条件下羊栖菜光合作用碳源获得机制比较(英文)

经济海洋褐藻羊栖菜(Hizikia fusiforme(Harv.)Okamura)低潮时常常周期性地暴露于空气中。为了认识这种海藻在潮汐循环背景下的光合特征,对其在高潮沉水和低潮干出不同条件下的光合作用碳素获得机制进行了比较。沉水时,羊栖菜主要利用海水中HCO_3~-作为外源无机碳源驱动光合作用;而在干出条件下,其光合作用的主要碳源为空气中的CO_2。在这两种不同环境条件下,光合作用与pH值的关系不同:沉水状态时,羊栖菜在高pH值(10.0)下光合活性很弱;而在干出条件下,羊栖菜在高pH值时仍有较高的光合活性。然而,光合作用无论是在沉水还是在干出条件下,对外源碳源的获得都表现出对胞外碳酸酐酶(CA)强烈的依赖性,并且其光合速率都受周围环境中无机碳源水平的限制。此外,在沉水和干出两种环境条件下,羊栖菜光合作用都表现出对氧气的敏感性。这表明,在羊栖菜中,依赖胞外CA的碳源获得机制不能使细胞内CO_2浓度提高到阻碍其光呼吸的程度。增加空气中或海水中无机碳的浓度,能促进羊栖菜的光合作用,进而增加这种海藻的水产养殖产量。     

Glypican and Biglycan in the Nuclei of Neurons and Glioma Cells: Presence of Functional Nuclear Localization Signals and Dynamic Changes in Glypican During the Cell Cycle

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target β-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican β-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.     

The ultrastructure of the marine blue green alga,Trichodesmium erythraeum,with special reference to the cell wall,gas vacuoles,and cylindrical bodies

Summary The marine blue green alga, Trichodesmium erythraeum, was studied with electron microscopy in an attempt to elucidate the structural basis for its rapid lysis when removed from its marine environment. In this connection, it was found that a thining of the electron-dense layer of the longitudinal wall at the site adjacent to transverse wall attachment was responsible for lysis. The underlying biochemical basis for this change has not been elucidated because of the extreme difficulties of maintaining and growing the alga in culture under defined conditions. Several other features of considerable interest also were found. Especially interesting is the very regular array of gas vacuoles in the form of a hollow cylinder which shields most of the photosynthetic system. It was suggested that the gas vacuoles might possibly function optically, having adaptive value in protecting the free-floating alga from excessive radiation. In addition, a detailed structure of the cylindrical bodies was presented, and its structure with the photosynthetic lamellae was compared. On the basis of sectoring to form fragments of double lamellar units from the cylindrical body which are identical in structure to the photosynthetic lamellae, it has been postulated that the cylindrical body may be the site of synthesis for the photosynthetic system in Trichodesmium erythraeum.     

Putative neurotransmitters in the retinae of three urodele species (Triturus alpestris,Salamandra salamandra,Pleurodeles waltli)

Summary The immunocytochemical localization of several substances with putative neurotransmitter or modulator properties was investigated in the retinae of three urodele species. Gamma-aminobutyric acid-like immunoreactive labelling appeared in different types of amacrine and horizontal cells. In addition, labelled fibres in the optic nerve were detected. It was not possible to determine whether these fibres were ganglion-cell axons or part of an efferent projection. Endogenous serotonin was found in several populations of amacrine cells including stratified and diffuse types. Glucagon-like immunoreactivity appeared in one bistratified amacrine cell type, and neurotensin-like immunoreactivity was detected in a single monostratified amacrine cell type. Metenkephalin-like-immunoreactive labelling was rare but found in several sublaminae of the inner plexiform layer. Thus each peptide-like-immunoreactive cell type makes up a distinct and unique population of cells and probably has a special functional role in retinal processing. There are striking similarities in the peptide-like immunoreactive patterns of Triturus alpestris and Necturus maculosus whereas in Ambystomatidae the peptide-like-immunoreactive systems appear to be differently organized. This supports the hypothesis that Salamandridae and Proteidae are more closely related to each other than to the Ambystomatidae.Abbreviations GABA gamma-aminobutyric acid - GCL ganglion cell layer - Glu glucagon - HRP horseradish peroxidase - INL inner nuclear layer - IPL inner plexiform layer - IR immunoreactive or immunoreactivity - M-enk metenkephalin - Neu neurotensin - OFL optic fibre layer - ONL outer nuclear layer - OPL outer plexiform layer - Ser serotonin This work forms part of the doctoral thesis of Gaby Gläsener, Faculty of Biology, Technical University of Darmstadt, Federal Republic of Germany. Supported by a research grant from the Deutsche Forschungsgemeinschaft (Hi 306/1-1)     

干出和沉水不同条件下羊栖菜光合作用碳源获得机制比较

经济海洋褐藻羊栖菜(Hizikia fusiforme(Harv.)Okamura)低潮时常常周期性地暴露于空气中.为了认识这种海藻在潮汐循环背景下的光合特征,对其在高潮沉水和低潮干出不同条件下的光合作用碳素获得机制进行了比较.沉水时,羊栖菜主要利用海水中HCO3-作为外源无机碳源驱动光合作用;而在干出条件下,其光合作用的主要碳源为空气中的CO2.在这两种不同环境条件下,光合作用与pH值的关系不同:沉水状态时,羊栖菜在高pH值(10.0)下光合活性很弱;而在干出条件下,羊栖菜在高pH值时仍有较高的光合活性.然而,光合作用无论是在沉水还是在干出条件下,对外源碳源的获得都表现出对胞外碳酸酐酶(CA)强烈的依赖性,并且其光合速率都受周围环境中无机碳源水平的限制.此外,在沉水和干出两种环境条件下,羊栖菜光合作用都表现出对氧气的敏感性.这表明,在羊栖菜中,依赖胞外CA的碳源获得机制不能使细胞内CO2浓度提高到阻碍其光呼吸的程度.增加空气中或海水中无机碳的浓度,能促进羊栖菜的光合作用,进而增加这种海藻的水产养殖产量.     

Disruption-free imaging by Raman spectroscopy reveals a chemical sphere with antifouling metabolites around macroalgae

Investigations of the surface chemistry of marine organisms are essential to understand their chemically mediated interactions with fouling organisms. In this context, the concentration of natural products in the immediate vicinity of algal surfaces, as well as their biological activity, are of particular importance. However, due to lack of appropriate methods, the distribution of compounds within the chemical sphere around marine algae is unknown. This study demonstrates the suitability of confocal resonance Raman microspectroscopy for the determination of metabolites around algal surfaces with a micrometer resolution. The spatial distribution of carotenoids in the diffusion boundary layer of the brown alga Fucus vesiculosus and the green alga Ulva sp. was determined using the disruption-free optical method. A gradient of carotenoids was determined within 0 to 150 μm from the surface of thealgae, thereby demonstrating the release of the non-polar metabolites involved in antifouling processes. Thedifferences in the carotenoid composition of the brown and green algae were reflected in the spectra. Resonance Raman microspectroscopy also allowed visualization of the lateral distribution of fucoxanthin on the algal surface and localization of concentration maxima within a 50 × 50 μm² area. The results from this work show clearly that established dipping techniques suitable for the extraction of the diffusion boundary layer of macroalgae only provide an average of the local strongly variable concentrations of metabolites on algal surfaces.     

Expression of different regional patterns of fibronectin immunoreactivity during mesoblast formation in the chick blastoderm

The appearance and distribution of the extracellular material glycoprotein, fibronectin, was investigated in gastrulating chick embryos using affinity-purified anti-human plasma fibronectin antibodies. Preservation of tissue structure and immunoreactivity was carried out by ethanol/acetic acid fixation or by formaldehyde/glutaraldehyde fixation. Using the former fixation method, fibronectin immunoreactivity was detected (1) at the ventral surface of the upper layer or epiblast, mainly anterior and lateral to Hensen's node, in regions where middle-layer or mesoblast cells are not yet present, and (2) sparsely in extracellular spaces of the deep layer. Using the latter fixation method, fibronectin immunoreactivity was, moreover, found at the entire ventral surface of the upper layer, i.e., also at the epithelial-mesenchymal interface, where a basement membrane was previously described. At the light microscope level, we could not detect significant immunoreactivity in the middle layer. Treatment of sections of ethanol-fixed blastoderms with testicular hyaluronidase before immunostaining for fibronectin partially demasked the antigenic sites of this glycoprotein at the epithelial-mesenchymal interface. The present report indicates that the different regional patterns of fibronectin immunoreactivity in the basement membrane of the upper layer are spatially and temporally correlated with migration and positioning of mesoblast cells. These regional patterns are probably due to differences in the composition of fibronectin-associated material such as chondroitin sulfate A and/or C proteoglycans, and/or hyaluronate, before and after mesoblast expansion, rather than to differences in the distribution of fibronectin itself. In this respect, it is noteworthy that the chemical composition of the basement membrane of an epithelium changes as mesenchyme cells migrate over it. The results also favor the idea that fibronectin is a structural component of the whole basement membrane which is used as a substrate for migration of mesenchymal cells.     

Ultrastructural immunocytochemical distribution of VIP-like immunoreactivity in dog ileum

The electron-immunocytochemical protein A-gold technique was employed to study the subcellular localization of vasoactive intestinal polypeptide-like material in dog ileum. The vasoactive intestinal polypeptide-like immunoreactivity was found within a population of large granular vesicles similar in structure in nerve varicosities of the myenteric plexus, the deep muscular plexus, the submucous plexus, the longitudinal muscular layer and the mucosa; none was found in nerve cell bodies. In the myenteric plexus, submucous plexus, the mucosa and the longitudinal muscular layer, varicosities containing similar large granular vesicles consistently remained unstained suggesting that within these plexuses morphologically indistinguishable by our technique large granular vesicles are not necessarily biochemically identical. In the deep muscular plexus, nearly all varicosities with large granular vesicles contained immunoreactivity for vasoactive intestinal polypeptide, but these varicosities often contained a few unstained large granular vesicles. This suggests that vasoactive intestinal polypeptide may share the same varicosity or the same vesicle with other neuropeptides present in this plexus (e.g., substance P or enkephalins) and that this plexus is a site where vasoactive intestinal polypeptide exerts its control over motility.     

Nitric oxide synthase immunoreactivity in the developing and adult human retina

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) from L-arginine. In this study, the cellular localization of neuronal NOS (nNOS) activity in the human retina since fetal development was examined by immunohistochemistry. No detectable staining in the fetal retina was present at 14 weeks of gestation (wg), the earliest age group examined. A centro-peripheral gradient of development of nNOS immunoreactivity was evident at 16–17 wg, with the midperipheral retina showing nNOS immunoreactivity in most of the cell types and the inner plexiform layer while the peripheral part demonstrated moderate immunoreactivity only in the ganglion cell layer and photoreceptor precursors. A transient increase in nNOS immunoreactivity in the ganglion cells and Müller cell endfeet between 18–19 and 24–25 wg was observed at the time when programmed cell death in the ganglion cell layer, loss of optic nerve fibres as well as increase in glutamate immunoreactivity and parvalbumin (a calcium binding protein) immunoreactivity in the ganglion cells was reported. These observations indicate that programmed cell death of ganglion cells in the retina may be linked to glutamate toxicity and NO activity, as also suggested by others in the retina and cerebral cortex. The presence of nNOS immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions, besides their involvement in photoreceptor function of transduction and information processing.     

Localization of the mGlu4a metabotropic glutamate receptor in rat cerebellar cortex

 The subcellular localization of the mGlu4a metabotropic glutamate receptor was investigated in rat cerebellum. At the light microscopical level, strong mGlu4a immunoreactivity was found in the molecular layer. A post-embedding immunogold method for electron microscopy revealed gold particles at the presynaptic sites of synapses made by parallel fiber terminals with dendritic spines of Purkinje cells. These observations support electrophysiological evidence indicating an autoreceptor function of mGlu4 receptors at these synapses. Accepted: 1 July 1997     

Immunocytochemical localization of monoamine oxidase type B in rat liver

We used an immunohistochemical method to examine the localization of monoamine oxidase type B (MAOB) in rat liver. At the light microscopic level, MAOB was highly expressed in rat liver. It was intense around portal area, and weak around central area. All the hepatocytes examined had MAOB immunoreactivity. For the first time, using a double-labeling immunofluorescence histochemical method for laser microscopy, we report that no MAOB is found in endothelial cells, hepatic stellate cells, or Kupffer's cells. When examined under transmission electron microscopy, MAOB was localized to the mitochondrial outer membrane of hepatocytes. No apparent localization of MAOB was found in the rough endoplasmic reticulum, the crystal membrane of mitochondria, the nuclear envelope, or the plasma membrane.