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  • Immunocytochemistry
  • Springer  (91)
  • 2020-2024
  • 1995-1999  (83)
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  • 2020-2024
  • 1995-1999  (83)
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  • 1
    ISSN: 1573-0832
    Keywords: ELISA ; Endodermis ; H. annosum ; Immunocytochemistry ; Root rot ; Vascular tissues
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Vascular disintegration mainly of medulla rays of spruce roots is of major significance in root rot disease of spruce caused byH. annosum. Using seedling roots as an experimental model, the possible routes and initial host reactions preceding invasion of vascular tissues was investigated. Transmission electron microscopy showed that penetration through the endodermis was an obvious route but not without host resistance. Using antibodies againstH. annosum hyphal materials, some labelling of vascular tissues remote from sites of fungal colonization suggest the release of fungal secretory products partly active in tissue disintegration. Similarly, intense labelling was also observed in severely colonized host tissues at late stages of infection. Strong labelling recorded at 3 d p.i. mainly on fungal hyphae and scant gold particles on invaded host tissues could imply that induction of host antifungal metabolites may have been a late event. A correlation was found between total antigenic material in root homogenates measured by ELISA, density of tissue labelling by immunocytochemistry and severity of disease symptoms. The importance of this in relation to diagnosis of biotic root rot diseases in the field is discussed.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Trees 13 (1999), S. 138-151 
    ISSN: 0931-1890
    Keywords: Key words Cytoskeleton ; Immunocytochemistry ; Model systems ; Populus ; Secondary vascular system
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract  Trees represent a, probably the, major component of the biosphere and have a unique place in the history of Mankind. One of their most fascinating features is the process of secondary growth which is effected principally by the secondary vascular system, the developmental continuum of secondary phloem, vascular cambium, and secondary xylem. However, for too long assumptions about the developmental biology of trees have had to be based upon studies of primary growth systems within annual, herbaceous species because study of the secondary vascular system had been largely ignored. Even when attempts are made to understand some of the most fundamental features of the secondary vascular system, such as xylogenesis, the current model system, isolated Zinnia mesophyll cells, is not entirely appropriate to the situation in the intact tree. Some deficiencies of the Zinnia system are discussed, and the advantages of the genus Populus as a model for study of the hardwood secondary vascular system are considered. Some of the new approaches which are poised to lead to significant advances in our knowledge of the cell bio-logy of the secondary vascular system of trees – spe-cifically of the cell wall, the plasmalemma, and the cytoskeleton – are discussed. The value of one of these new techniques – immunocytochemistry – is demonstrated by a consideration of recent work on the role of the cytoskeleton in the hardwood secondary vascular system.
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  • 3
    ISSN: 1432-1424
    Keywords: Water channels ; Vasopressin ; Rat kidney ; Immunocytochemistry ; Microtubules ; Cell polarity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Aquaporin 2 is a collecting duct water channel that is located in apical vesicles and in the apical plasma membrane of collecting duct principal cells. It shares 42% identity with the proximal tubule/thin descending limb water channel, CHIP28. The present study was aimed at addressing three questions concerning the location and behavior of the AQP2 protein under different conditions. First, does the AQP2 channel relocate to the apical membrane after vasopressin treatment? Our results show that AQP2 is diffusely distributed in cytoplasmic vesicles in collecting duct principal cells of homozygous Brattleboro rats that lack vasopressin. In rats injected with exogenous vasopressin, however, AQP2 became concentrated in the apical plasma membrane of principal cells, as determined by immunofluorescence and immunogold electron microscopy. This behavior is consistent with the idea that AQP2 is the vasopressin-sensitive water channel. Second, is the cellular location of AQP2 modified by microtubule disruption? In normal rats, AQP2 has a mainly apical and subapical location in principal cells, but in colchicine-treated rats, it is distributed on vesicles that are scattered throughout the entire cytoplasm. This is consistent with the dependence on microtubules of apical protein targeting in many cell types, and explains the inhibitory effect of microtubule disruption on the hydroosmotic response to vasopressin in sensitive epithelia, including the collecting duct. Third, is AQP2 present in neonatal rat kidneys? We show that AQP2 is abundant in principal cells from neonatal rats at all days after birth. The detection of AQP2 in early neonatal kidneys indicates that a lack of this protein is not responsible for the relatively weak urinary concentrating response to vasopressin seen in neonatal rats.
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  • 4
    ISSN: 1432-0878
    Keywords: Rat posterior pituitary ; Neurophysin-vasopressinergic and neurophysin-oxytocinergic fibres ; Immunocytochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary By means of the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the electron microscopic level, it was demonstrated that the hormones of the neural lobe of the rat hypophysis are located in separate neurophysin-vasopressinergic and neurophysin-oxytocinergic nerve fibres. These observations confirm the results of our previous immunocytochemical studies at the light microscopic level.
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  • 5
    ISSN: 1432-0878
    Keywords: Pituitary gland ; Cell types ; Estrogen ; Autoradiography ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Dry-mount autoradiography was combined with peroxidase immunocytochemistry to examine estrogen uptake in four pituitary cell types. Quantification by silver grain counts was used to compare 3H-estradiol uptake in nuclei of pituitary cells 60 min after i.v. injection into short-term (control) and long-term ovariectomized and in long-term thyroidectomized rats. Under all three hormonal states, the order of labeling intensity was: gonadotropes 〉 somatotropes 〉 lactotropes 〉 thyrotropes. Long-term ovariectomy caused a significant increase in estrogen uptake of gonadotropes, somatotropes and lactotropes, while uptake in thyrotropes decreased. Long-term thyroidectomy decreased uptake in somatotropes, lactotropes and thyrotropes while gonadotropes remained unchanged.
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  • 6
    ISSN: 1432-0878
    Keywords: Nerve growth factor ; Submandibular gland mice ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Nerve growth factor (NGF) was localized in the submandibular gland of adult male mice by a direct immunocytochemical method using highly purified antibodies against NGF coupled to horseradish peroxidase. In light microscopic sections the reaction product was entirely confined to the cells of the secretory tubules. The acinar part of the gland was free of reaction product. This finding was confirmed by electron microscopy. Within the cells NGF was localized exclusively in the apical secretory granules. No reaction was observed in the rough endoplasmic reticulum, the Golgi region or in the granules of the basal part of the cells. This observation favours the assumption that NGF is derived from a precursor molecule and that the precursor is transformed into immunologically active NGF within the secretory granules during their transport from the basal to the apical part of the tubular cells. Stimulation of the submandibular gland with carbachol (2 mg/kg) led to a massive release of the content of the secretory granules, including NGF, into the salivary duct.
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  • 7
    ISSN: 1432-0878
    Keywords: Luteinizing hormone-releasing hormone ; Median eminence ; Electron microscopy ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary LH-RH was localized at the ultrastructural level in axons and nerve terminals of the median eminence of the male guinea pig. LH-RH positive neuronal profiles were most concentrated in the medial-dorsal aspect of the infundibular stalk and in the post-infundibular median eminence at the level immediately following separation of the stalk from the base of the brain. LH-RH containing axon profiles were most abundant in the palisade zone; nerve terminals in contact with the hypophysial portal vasculature were relatively rare. The hormone was present within granules that measured 900–1,200 Å in axons of the palisade zone and 400–800 Å in nerve terminals abutting on the portal plexus. The differently sized granules represent heterogeneous populations.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 170 (1976), S. 263-273 
    ISSN: 1432-0878
    Keywords: Fetal pituitary ; Cell differentiation ; Immunocytochemistry ; Tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Adenohypophysial primordia of rat embryos at 13 to 15 days gestation were cultured in Parker 199 synthetic medium for 2 to 11 days. At the end of the culture period their fine structure and the presence of immunoreactive trophic hormones using the peroxidase-labeled antibody technique were investigated. The degree of differentiation in the glands depends largely on the age of the embryos furnishing the explants. Cultured pituitaries explanted on the 13th day of gestation contain only ACTH-positive cells and about 15% of the cells are granular. The granules are 50–100 nm in diameter in some cells, while in other cells they range from 50 to 200 nm. In cultivated adenohypophysial primordia of embryos on the 15th day of intrauterine life ACTH, prolactin, LH and TSH cells are evident, but only the same two kinds of granular cells can be observed with the electron microscope. The extent of cytodifferentiation in the glands explanted on the 14th day of gestation is intermediate between the two other groups. The data suggest that the fetal rat pituitary has the capacity of self-differentiation but to a lesser extent than that of the in situ hypophysis.
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  • 9
    ISSN: 1432-0878
    Keywords: Amphibian hypothalamus ; Vasotocinergic and mesotocinergic neurons ; Neurophysins ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Using the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the light microscopic level, it was demonstrated that, in the amphibian magnocellular hypothalamo-hypophysial neurosecretory system, vasotocin and mesotocin are synthesized in separate neurons. A tendency to preferential location of the two kinds of neuronal perikarya is described. The neurosecretory perikarya are the origin of separate vasotocinergic and mesotocinergic axons. In the neural lobe, the pattern of distribution of the two types of axons is different. The coarse ventricular “dendrites” of both kinds of neurons are hormone-containing processes. Staining with anti-bovine neurophysin I serum suggested that the vasotocinergic and the mesotocinergic neurons synthesize different neurophysins.
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  • 10
    ISSN: 1432-0878
    Keywords: Amphibian posterior pituitary ; Vasotocinergic and mesotocinergic fibres ; Immunocytochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Using the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the electron microscopic level, it was demonstrated that the hormones of the posterior pituitary of Rana temporaria are located in separate vasotocinergic and mesotocinergic nerve fibres. This observation confirms the results of our previous immunocytochemical studies at the light microscopic level.
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  • 11
    Electronic Resource
    Electronic Resource
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    Cell & tissue research 175 (1976), S. 165-181 
    ISSN: 1432-0878
    Keywords: Pig ; Neurophysin-I ; Neurophysin-II ; Immunocytochemistry ; Specific neurophysin neurosecretory systems
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Antibodies raised against porcine neurophysin-I and porcine neurophysin-II using an injection regime in rabbits over a short time period, were used to localize neurophysin-I and neurophysin-II in hypothalamic neurosecretory elements of the domestic pig. In transverse section, neurophysin-II containing cells were more abundant in the dorsal medial region of the rostral supraoptic nucleus (SON) as compared with the distribution of neurophysin-I neurons. The main bulk of the cells of the SON were heavily stained for neurophysin-I with neurophysin-II containing cells positioned dorsal from the edge of the optic chiasma. Neurosecretory cells of the SON as seen in sagittal section also showed a differential staining for neurophysins-I and -II. Rostral regions of the pig paraventricular nucleus (PVN) contained magnocellular elements near the third ventricle which were stained predominantly for neurophysin-II. In regions corresponding to the caudal PVN there appeared two populations of neurosecretory neurons: (a) an area of cells adjacent to the third ventricle which contained neurophysin-II antigen and (b) a group of densely populated cells in the dorsal-lateral region which was stained for neurophysin-I. The results support the existence in the pig of at least two distinct populations of neurosecretory neurons corresponding to the neurophysin-I and neurophysin-II neurosecretory system.
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  • 12
    ISSN: 1432-0878
    Keywords: Oxytocin ; Neurophysin ; Vasotocin ; Mesotocin ; Suprachiasmatic nucleus ; Medial nucleus of the infundibular recess ; Immunocytochemistry ; Natrix maura (Serpentes) ; Mauremys caspica (Chelonia)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The probable presence of oxytocin in the hypothalamo-hypophysial system of two reptilian species, the snake Natrix maura and the turtle Mauremys caspica, was re-investigated. A high-pressure liquid chromatographic analysis of the turtle neural lobe revealed the existence of vasotocin, mesotocin, and a third compound co-eluting with oxytocin. Brains from both species were fixed by vascular perfusion with Bouin's fluid. Adjacent paraffin sections were immunostained using antisera against the following substances: (1) bovine oxytocin-neurophysin; (2) a mixture of bovine oxytocin-neurophysin and vasopressin-neurophysin; (3) dogfish neurophysins; (4) oxytocin; (5) arginine-vasotocin; (6) mesotocin; (7) somatostatin. Immunoreactivity against oxytocin was found in parvocellular neurons of the snake suprachiasmatic nucleus and cerebrospinal-fluid contacting neurons of the medial nucleus of the infundibular recess of both species, the latter immunoreactivity being much more conspicuous in the turtle. Numerous fibers containing immunoreactive oxytocin extended between the medial nucleus of the infundibular recess, and the internal region of the medium eminence and the neural lobe. The oxytocin-immunoreactivity in all locations was completely abolished by preabsorption of the anti-oxytocin serum with three different oxytocin preparations. None of the neurons of the suprachiasmatic and medial nucleus of the infundibular recess, including the oxytocin-immunoreactive elements, reacted with either the antineurophysin sera used, or the anti-vasotocin or anti-mesotocin antibodies. The possible existence of a reptilian oxytocin-neurophysin is discussed. The alternative that, in the reptilian hypothalamus, neurons synthesize a compound closely related to, but different from oxytocin is also considered.
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  • 13
    ISSN: 1432-0878
    Keywords: Pituitary gland ; Gonadotropin ; Subunits ; Gonadotropes ; Immunocytochemistry ; Immunoblotting ; Oncorhynchus mykiss (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Salmon gonadotropin (GTH II) is a heterodimeric glycoprotein hormone (α and IIβ subunits), serving as a maturational GTH, and is produced in a specific gonadotropic cell-type (GTH II-cells) containing small granules and large globules. In trout GTH II-cells, double immunolabeling for the α- and IIβ-subunits shows that colocalization of the α- and IIβ-immunolabeling is confined to the small granules, indicating storage of functional GTH II. On the other hand, α-immunolabeling is absent in the large globules, even though IIβ labeling is abundant throughout the period of seasonal gametogenesis. The α-specific antiserum recognizes the intact α-subunit as well as the reduced and deglycosylated α-subunits by immunoblotting. These results indicate that an accumulation of the IIβ-subunit is specifically generated in the large globules of these cells. In fact, with sexual maturity, the quantity of IIβ-subunits becomes elevated in the trout pituitary due to a marked increase in GTH II-cells containing many large globules. However, the derivation and function of the large globules and the fate of their contained IIβ-subunits remains unknown.
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  • 14
    ISSN: 1432-0878
    Keywords: Trans-differentiation ; Proliferation ; Bromodeoxyuridine ; Immunocytochemistry ; Regeneration ; Ciona intestinalis (Tunicata)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract In this study, we use three monoclonal antibodies that recognise antigens present in the central nervous system of the ascidian Ciona intestinalis to study regeneration and post-metamorphic development of the neural ganglion. We have also used bromodeoxyuridine labelling to study generation of the neuronal precursor cells. The first antibody, CiN 1, recognises all neurones in the ganglion, whereas the second, CiN 2, recognises only a subpopulation of the large cortical neurones. Western blotting studies show that CiN 2 recognises two membrane-bound glycoproteins of apparent Mr 129 and 100 kDa. CiN 1 is not reactive on Western blots. Immunocytochemical studies with these antibodies show that CiN 1-immunoreactive neurone-like cells are present at the site of regeneration as early as 5–7 days post-ablation, a sub-population of CiN 2-immunoreactive cells being detected by 9–12 days post-ablation. The third antibody, ECM 1, stains extracellular matrix components and recognises two diffuse bands on Western blots of whole-body and ganglion homogenates. The temporal and spatial pattern of appearance of CiN 1 and CiN 2 immunoreactivity both during post-metamorphic development and in regeneration occurs in the same sequence in both processes. Studies with bromodeoxyuridine show labelled nuclei in some neurones in the regenerating ganglion. Plausibly these originate from the dorsal strand, an epithelial tube that reforms by cell proliferation during the initial phases of regeneration. A second population of cells, the large cortical neurones, do not incorporate bromodeoxyuridine and thus must have been born prior to the onset of regeneration. This latter finding indicates a mechanism involving trans-differentiation of other cell types or differentiation of long-lived totipotent stem cells.
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  • 15
    Electronic Resource
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    Cell & tissue research 280 (1995), S. 541-548 
    ISSN: 1432-0878
    Keywords: Key words: Musle ; striated ; skeletal ; Regeneration ; Myosin ; Immunocytochemistry ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Indirect immunofluorescence was used to localize embryonic myosin heavy chains in soleus, adductor longus, tibialis anterior, plantaris, and extensor digitorum longus muscles of 6-month-old rats. A monoclonal antibody (2B6), specifically recognizing rat embryonic myosin, was applied to unfixed, transverse, frozen sections. The number of embryonic myosin-positive (EMP) extrafusal fibers was expressed as a percentage of the total number of fibers. EMP extrafusal fibers were only seen in the soleus and adductor longus muscles, both postural muscles. Approximately 1% of the soleus muscle fibers appeared positively stained for embryonic myosin. The majority of such fibers had a small diameter (〈500 μ2), appeared intensely fluorescent, and typically contained central nuclei. Re-expression of embryonic myosin due to spontaneous fiber denervation is not a likely factor in this study, since alpha-bungarotoxin and N-CAM localization were restricted to the motor end-plate region of EMP fibers. Since embryonic myosin was shown to disappear in all normal-sized myofibers by 2 to 3 months of age, the results suggest that the EMP extrafusal fibers seen in postural muscles of 6 to 12-month-old animals are regenerating myofibers. We speculate that a small number of muscle fibers may be regenerating in normal, adult postural muscles, in response to fiber damage possibly caused by excessive recruitment or overloading.
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  • 16
    ISSN: 1432-0878
    Keywords: Key words: Melanin-concentrating hormone ; Immunocytochemistry ; Development ; ontogenetic ; Sparus auratus (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The development of the hypothalamic melanin-concentrating hormone (MCH) system of the teleost Sparus auratus has been studied by immunocytochemistry using an anti-salmon MCH serum. Immunoreactive perikarya and fibers are found in embryos, larvae, and juvenile specimens. In juveniles, most labeled neurons are present in the nucleus lateralis tuberis; some are dispersed in the nucleus recessus lateralis and nucleus periventricularis posterior. From the nucleus lateralis tuberis, MCH neurons project a conspicuous tract of fibers to the ventral hypothalamus; this penetrates the pituitary stalk and reaches the neurohypophysis. Most fibers end close to the cells of the pars intermedia, and some reach the adenohypophysial rostral pars distalis. Immunoreactive fibers can also be seen in extrahypophysial localizations, such as the preoptic region and the nucleus sacci vasculosi. In embryos, MCH-immunoreactive neurons first appear at 36 h post-fertilization in the ventrolateral margin of the developing hypothalamus. In larvae, at 4 days post-hatching, perikarya can be observed in the ventrolateral border of the hypothalamus and in the mid-hypothalamus, near the ventricle. At 26 days post-hatching, MCH perikarya are restricted to the nucleus lateralis tuberis. The neurohypophysis possesses MCH-immunoreactive fibers from the second day post-hatching. The results indicate that MCH plays a role in larval development with respect to skin melanophores and cells that secrete melanocyte-stimulating hormone.
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  • 17
    ISSN: 1432-0878
    Keywords: Key words: Neuropeptide Y ; Gastroenteropancreatic (GEP) endocrine system ; Development ; ontogenetic ; Vitellointestinal duct ; Pancreas ; exocrine ; Pancreas ; endocrine ; Immunocytochemistry ; Scyliorhinus torazame (Elasmobranchii)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. This immunocytochemical study was carried out to elucidate the ontogenetic development of neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system of the cloudy dogfish, Scyliorhinus torazame. Immunostained cells first appeared in the pancreas of the embryo at the 15-mm stage, and were also detected in the vitellointestinal duct of the yolk stalk at the 20-mm stage. These cells were polymorphic, with occasional processes that were sometimes directed toward the vascular wall or into the cavity of the vitellointestinal duct. At the 34-mm stage, immunostained cells could also be found in the proximal part of the spiral intestine and, by the 74-mm stage, immunopositive cells were present in the gastric mucosa. In the gut and pancreas, the cells gradually increased in number with development, whereas in the vitellointestinal duct and internal yolk sac, they decreased and seemed to disappear following hatching. Thus, in juveniles, the distribution of the neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system had attained that of adults. Electron-microscopic immunocytochemistry demonstrated that, in the labeled cells of the vitellointestinal duct, the neuropeptide Y-like antigen was located in cytoplasmic granules, as in the cells of the gut and pancreas.
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  • 18
    ISSN: 1432-0878
    Keywords: Key words: Esophagus ; Epithelial cells ; Intestinal lectin ; L-36 ; RI-H fragment ; Immunocytochemistry ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Using an affinity purified antibody raised against the RI-H fragment of rat intestinal lectin L-36, the latter protein has been identified within the esophageal epithelium by means of ultracryotomy followed by immunogold labeling. The epithelium consists of 4 morphologically distinct cell-types, namely, the basal, spiny, granular and squamous cells, and each of these exhibits a different immunolabeling pattern. The basal cells form a layer on the basal lamina, and in these a diffuse cytoplasmic staining is observed. This basal cell layer is overlaid by spiny cells that extend many cell processes into wide intercellular spaces. In these cells, immunogold particles are found only on small granular inclusions consisting of an electron-lucent homogeneous substance. The granular cells form a third layer over the spiny cells, and are characterized by a number of large granular inclusions with an electron-dense core rimmed by a less electron-dense substance. Immunogold labeling is found on these granules, both on the core and peripheral region. Squamous cell-types constitute the most superficial layer of the epithelium. They are without granular inclusions, and immunogold labeling is confined to the cytoplasmic surface of the thickened plasma membrane. These findings suggest that L-36 is produced in the basal cells as free cytosolic protein, then becomes progressively aggregated into the granular inclusions of the spiny and granular cells, and is eventually transferred onto the cytoplasmic surface of the squamous cell plasma membrane where it may interact with complementary glycoconjugate(s) located at this site. The membrane lining substance thus formed may play a role in stabilizing the squamous cell membranes, thereby maintaining the structural integrity of the epithelium against mechanical stress coming from the esophageal lumen.
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  • 19
    ISSN: 1432-0878
    Keywords: Key words: Epididymis ; Efferent ducts ; Cell culture ; Immunocytochemistry ; Immunoprecipitation ; Man
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The way in which the human epididymis modifies spermatozoa during their sojourn in this structure might be clarified by knowledge of the nature of its secretions. We have examined the presence of several lysosomal hydrolases in human epididymal tissue and fluids, and their synthesis and secretion by monolayer cultures. Tissues were obtained from men undergoing orchidectomy for prostatic carcinoma. The enzymes cathepsin D and acid α-glucosidase were localised in the lysosomes of epithelial cells from the corpus epididymidis, by an immunocytochemical technique. Cathepsin D was also found in epithelial cells of the efferent ducts within lysosomes, apical vesicles and multivesicular bodies. No immunolocalisation of acid glucosidase in the efferent ducts or on the microvilli of the corpus was demonstrable. Cathepsin D, β-hexosaminidase (N-acetylglucosaminidase) and α-glucosidase were measurable in the luminal fluid from the human corpus epididymidis; β-hexosaminidase was secreted into the culture medium by confluent monolayers of epididymal and efferent duct cells. Immunoprecipitation of cell extracts and culture medium of these cultures incubated with 35S-methionine revealed that the precursors of cathepsin D and β-hexosaminidase were synthesized and secreted by such monolayers. Thus, active lytic enzymes are secreted by the human epididymis and could modify sperm membranes.
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  • 20
    ISSN: 1432-0878
    Keywords: Compound eye ; Photoreceptor cells ; Ion pumps ; Polarity ; Immunocytochemistry ; Manduca sexta (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Immunohistochemical and physiological studies on various insect photoreceptors have demonstrated that the Na,K-ATPase (sodium pump) is restricted to the nonreceptive nonmicrovillar area of the plasma membrane. Here, we examined the distribution of the Na,K-ATPase in photoreceptor cells of the superposition-type compound eye in the moth Manduca sexta. Using immunofluorescent and immunogold cytochemistry, we show that the Na,K-ATPase is localized to both the nonmicrovillar and the microvillar parts of the plasma membrane. Manduca photoreceptors thus deviate from the common concept that the sodium pump and the molecular components of the photoreceptive machinery reside on different domains of the plasma membrane.
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  • 21
    ISSN: 1432-0878
    Keywords: Neurohemal areas ; Neuropeptides ; Monoamines ; Immunocytochemistry ; Nervous system, insect ; Gryllus bimaculatus (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The morphology and position of putative neurohemal areas in the peripheral nervous system (ventral nerve cord and retrocerebral complex) of the cricket Gryllus bimaculatus are described. By using antisera to the amines dopamine, histamine, octopamine, and serotonin, and the neuropeptides crustacean cardioactive peptide, FMRFamide, leucokinin 1, and proctolin, an extensive system of varicose fibers has been detected throughout the nerves of all neuromeres, except for nerve 2 of the prothoracic ganglion. Immunoreactive varicose fibers occur mainly in a superficial position at the neurilemma, indicating neurosecretory storage and release of neuroactive compounds. The varicose fibers are projections from central or peripheral neurons that may extend over more than one segment. The peripheral fiber varicosities show segment-specific arrangements for each of the substances investigated. Immunoreactivity to histamine and octopamine is mainly found in the nerves of abdominal segments, whereas serotonin immunoreactivity is concentrated in subesophageal and terminal ganglion nerves. Immunoreactivity to FMRFamide and crustacean cardioactive peptide is widespread throughout all segments. Structures immunoreactive to leucokinin 1 are present in abdominal nerves, and proctolin immunostaining is found in the terminal ganglion and thoracic nerves. Codistribution of peripheral varicose fiber plexuses is regularly seen for amines and peptides, whereas the colocalization of substances in neurons has not been detected for any of the neuroactive compounds investigated. The varicose fiber system is regarded as complementary to the classical neurohemal organs.
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  • 22
    ISSN: 1432-0878
    Keywords: Interleukin ; Stellate reticulum ; Immunocytochemistry ; Epidermal growth factor ; Interleukin-1 receptor type I messenger RNA ; Tooth eruption ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Immunolocalization of interleukin-1α in the first mandibular molars of rats from day 0–12 postnatally showed that the protein was localized in the epithelial stellate reticulum adjacent to the dental follicle. Staining of the stellate reticulum was most prominent in the early days postnatally and was absent by postnatal day 11. Injection of epidermal growth factor into rats at day 0 greatly increased the intensity of the staining for interleukin-1α in the stellate reticulum. Epidermal growth factor (EGF) enhanced the gene expression of interleukin-1α in stellate reticulum cells in vitro, and this study suggests there is enhanced translation of interleukin-1α messenger RNA in the stellate reticulum following EGF injection. In turn, the interleukin-1α may exert its effect on the dental follicle cells adjacent to the stellate reticulum because EGF also enhanced expression of the interleukin-1 receptor type I messenger RNA in cultured dental follicle cells as well as enhancing its expression in vivo. In view of the fact that injection of EGF will stimulate precocious eruption of teeth, its stimulus of interleukin-1α synthesis in the stellate reticulum may be the mechanism by which EGF initiates a cascade of molecular events to signal the onset of tooth eruption.
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  • 23
    ISSN: 1432-0878
    Keywords: Key words: Galectin ; β-Galactoside-binding lectin ; Human ; Skin ; Immunocytochemistry ; Immunohistochemistry ; Hybridization ; in situ ; Langerhans cell ; Man
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The localization of an endogenous 14-kDa β-galactoside-binding lectin (galectin) and its pattern of gene expression were examined in normal human skin by light- and electron microscopy. Under the light microscope, immunostaining of 14-kDa galectin was observed in the cell membrane of cells in the basal and spinous layers of the epidermis. Galectin was also found in the Langerhans cells, as shown by double labeling using anti-14-kDa galectin and anti-CD1a antibodi es. In the dermis, immunostaining for the 14-kDa galectin was positive in the extracellular matrix and fibroblasts. At the electron-microscopic level of resolution, galectin was located primarily along the plasma membrane of keratinocytes, and in both the cytoplasm and nucleus of Langerhans cells in the epidermis, whereas in the dermis it was detected in the extracellular matrix and in both the nucleus and cytoplasm of fibroblasts. The gene expression of 14-kDa galectin was visualized by the HRP-staining me thod following in situ hybridization techniques. The expression was detected in the cytoplasm of cells in the basal and spinous layers of the epidermis; whereas, in the dermis, it was detected in the cytoplasm of fibroblasts. Moreover, SDS-polyacrylamide gel electrophoresis and lectin-blot analysis revealed that this galectin bound to glycoproteins of approximately 17, 62, and 72 kDa in the epidermis and to those of 29, 54, and 220 kDa in the dermis. The present study indicates that 1) normal human skin produces the β-galactoside-binding 14-kDa galectin, and 2) this galectin is located in both the epidermis, particularly in the keratinocytes and Langerhans cells, and in the dermis. These results suggest that galectin is important for cell-cell contact and/or adhesion in the epidermis and for cell-extracellular matrix interaction in the dermis.
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  • 24
    ISSN: 1432-0878
    Keywords: Galectin ; β-Galactoside-binding lectin ; Human ; Skin ; Immunocytochemistry ; Immunohistochemistry ; Hybridization, in situ ; Langerhans cell ; Man
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The localization of an endogenous 14-kDa β-galactoside-binding lectin (galectin) and its pattern of gene expression were examined in normal human skin by light- and electron microscopy. Under the light microscope, immunostaining of 14-kDa galectin was observed in the cell membrane of cells in the basal and spinous layers of the epidermis. Galectin was also found in the Langerhans cells, as shown by double labeling using anti-14-kDa galectin and anti-CD1a antibodies. In the dermis, immunostaining for the 14-kDa galectin was positive in the extracellular matrix and fibroblasts. At the electron-microscopic level of resolution, galectin was located primarily along the plasma membrane of keratinocytes, and in both the cytoplasm and nucleus of Langerhans cells in the epidermis, whereas in the dermis it was detected in the extracellular matrix and in both the nucleus and cytoplasm of fibroblasts. The gene expression of 14-kDa galectin was visualized by the HRP-staining method following in situ hybridization techniques. The expression was detected in the cytoplasm of cells in the basal and spinous layers of the epidermis; whereas, in the dermis, it was detected in the cytoplasm of fibroblasts. Moreover, SDS-polyacrylamide gel electrophoresis and lectin-blot analysis revealed that this galectin bound to glycoproteins of approximately 17, 62, and 72 kDa in the epidermis and to those of 29, 54, and 220 kDa in the dermis. The present study indicates that 1) normal human skin produces the β-galactoside-binding 14-kDa galectin, and 2) this galectin is located in both the epidermis, particularly in the keratinocytes and Langerhans cells, and in the dermis. These results suggest that galectin is important for cell-cell contact and/or adhesion in the epidermis and for cell-extracellular matrix interaction in the dermis.
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  • 25
    ISSN: 1432-0878
    Keywords: Defence mechanisms ; Encapsulation ; Granulocytes ; Immunocytochemistry ; Parasitism ; Perkinsus sp. (Protozoa) ; Tapes semidecussatus (Mollusca)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Parasitosis by the trophozoite protozoan Perkinsus sp. (Apicomplexa, Perkinsea) induces in the gill filaments of the clam Tapes semidecussatus (Mollusca, Bivalvia) a cellular reaction, which is constituted by infiltrated granulocytes. This cellular reaction has characteristics of those of a holocrine gland, since the parasites are encapsulated by the secretion product of the granulocytes after cell death. An enriched fraction of prezoosporangia and their associated capsule was obtained after culture of the parasitized gills in fluid thioglycollate medium. Specific polypeptides from this fraction were separated by SDS-PAGE and isolated for rabbit immunizations. The serum obtained against an Mr 225 kDa polypeptide, revealed its exclusive localization in the capsule and in the granules of the infiltrated granulocytes, thus indicating that this polypeptide is synthesized by these cells and secreted, in a polarized way, around the trophozoites resulting in their encapsulation. Selective deglycosylation of the polypeptide, by Endo H and alkaline β-elimination, did not show an effect on its molecular weight or antibody recognition. Furthermore, the absence of the 225 kDa band in the Western-blots of non-parasitized gills indicated the specific association of this polypeptide with the parasitosis. Finally, this is the first tissue-specific factor described in molluscs in relation to defence mechanisms.
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  • 26
    ISSN: 1432-0878
    Keywords: Oocyte ; Yolk granules ; Ribonuclease ; Immunocytochemistry ; Bullfrog, Rana catesbeiana (Anura)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.
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  • 27
    ISSN: 1432-0878
    Keywords: Esophagus ; Epithelial cells ; Intestinal lectin, L-36 ; RI-H fragment ; Immunocytochemistry ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Using an affinity purified antibody raised against the RI-H fragment of rat intestinal lectin L-36, the latter protein has been identified within the esophageal epithelium by means of ultracryotomy followed by immunogold labeling. The epithelium consists of 4 morphologically distinct cell-types, namely, the basal, spiny, granular and squamous cells, and each of these exhibits a different immunolabeling pattern. The basal cells form a layer on the basal lamina, and in these a diffuse cytoplasmic staining is observed. This basal cell layer is overlaid by spiny cells that extend many cell processes into wide intercellular spaces. In these cells, immunogold particles are found only on small granular inclusions consisting of an electron-lucent homogeneous substance. The granular cells from a third layer over the spiny cells, and are characterized by a number of large granular inclusions with an electron-dense core rimmed by a less electron-dense substance. Immunogold labeling is found on these granules, both on the core and peripheral region. Squamous cell-types constitute the most superficial layer of the epithelium. They are without granular inclusions, and immunogold labeling is confined to the cytoplasmic surface of the thickened plasma membrane. These findings suggest that L-36 is produced in the basal cells as free cytosolic protein, then becomes progressively aggregated into the granular inclusions of the spiny and granular cells, and is eventually transferred onto the cytoplasmic surface of the squamous cell plasma membrane where it may interact with complementary glycoconjugate(s) located at this site. The membrane lining substance thus formed may play a role in stabilizing the squamous cell membranes, thereby maintaining the structural integrity of the epithelium against mechanical stress coming from the esophageal lumen.
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  • 28
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    Cell & tissue research 281 (1995), S. 101-108 
    ISSN: 1432-0878
    Keywords: Somite ; Intergin ; Extracellular matrix, structures ; Embryo ; Laminin ; Immunocytochemistry ; Macaca fascicularis (Primates)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The distribution of integrin subunits α6 and β1, and the α6β1 integrin ligand, laminin, was examined during somitogenesis in developmental stages 11, 13, and 16 in the long-tailed macaque, using peroxidase immunocytochemistry. Within differentiating somites in stage 11, α6 expression was observed in the sclerotome, basal surface of dermamyotomal cells adjacent to the basal lamina and on scattered cells throughout the dermamyotome. In further advanced somites in stages 13 and 16, α6 immunoreactivity become restricted to the myotome, α6 was expressed on mesenchymal core cells within the myocele of undifferentiated epitheliod somites and the ventromedial wall of somites commencing differentiation at each stage. β1 distribution resembled that of α6 in stage 11 somitic tissue, however, it remained present on myotome and sclerotome cells in the later stages, and was also expressed on dermatomal cells in stage 16. Laminin immunoreactivity, while more intense and prevalent than α6 and β1 in each stage examined, occurred on the same somite cell populations as the 2 integrin subunits. These results show a defined distribution of α6 on somitic tissue, and suggest this integrin is involved in somite differentiation. They also support a possible role for α6 in myoblast formation and migration. Overlapping of β1 and laminin immunoreactivity with that of α6 further suggests that α6 paris with β1 as a functional heterodimer for laminin in defined somitic regions.
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  • 29
    ISSN: 1432-0878
    Keywords: Key words: Gap junction ; Intercellular junction ; Insect ; Arthropod ; Immunocytochemistry ; Heliothis virescens (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Gap junction-enriched fractions were prepared from larvae of the tobacco budworm Heliothis virescens using the NaOH procedure in the presence or absence of protease inhibitors and were analyzed by SDS-PAGE, immunoblotting and EM immunocytochemistry. Protease inhibitor fractions contained a 48-kDa protein in addition to the ∼10 proteins in fractions with and without inhibitors. Three polyclonal antibodies were used as probes for gap junction plaques and proteins: R16, against an ∼40-kDa candidate gap junction protein from Drosophila melanogaster; R17, against the 40-kDa candidate gap junction protein from H. virescens; and R18AP, an affinity purified antibody against a consensus sequence of N-terminal amino acids 2–21 of the H. virescens 40-kDa protein. R16, R17, and R18AP stain the 40- and 48-kDa proteins, R16 and R18AP stain a 64-kDa protein, and R16 stains an ∼30-kDa protein in the absence of inhibitors. Inclusion of protease inhibitors had no effect on gap junction ultrastructure. R16 and R17 label gap junction plaques in crude membrane and NaOH fractions, whereas R18AP exhibits only a low level of reactivity with gap junctions in crude membrane fractions and none with gap junctions in NaOH fractions. The results show that the 30-, 40-, 48- and 64-kDa proteins are immunologically related and are associated with gap junctions in H. virescens, the N-terminus of the 40-kDa protein is relatively inaccessible or easily lost, and the 48-kDa protein is protease-sensitive.
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  • 30
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    Cell & tissue research 281 (1995), S. 507-515 
    ISSN: 1432-0878
    Keywords: Microtubules ; Isoforms ; Nervous system ; Locomotion ; Cilia ; Immunocytochemistry ; Western blotting ; Lymnaea stagnalis (Mollusca)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Immunocytochemistry and Western blotting techniques demonstrated that the nervous system and foot of the pond snail Lymnaea stagnalis are rich sources of tubulin, which can be extracted and assembled in vitro in the presence of taxol. Various broad-spectrum antibodies raised against α-tubulin and β-tubulin yielded qualitatively similar results. One monoclonal antibody to trypanosome α-tubulin, however, labelled α-tubulin more strongly on both probed sections and Western blots. Cytochemistry and immunoblotting revealed that tyrosinated tubulin constitutes a large proportion of total α-tubulin in locomotor cilia of the foot and in axons of the nervous system. Detyrosinated tubulin also appeared to be abundant in the foot cilia but only a very faint band of detyrosinated tubulin was found on protein blots extracted from the central ganglia, and staining was barely detectable in central ganglia or peripheral nerves. Similarly, acetylated tubulin appeared to be abundant in foot cilia, but Western blotting indicated only low levels of acetylated tubulin in the nervous system. Immunocytochemistry indicated that, while most neurons possessed little or no acetylated tubulin, a small number of axons contained significant amounts of this isoform. Thus, while a large amount of tubulin was expected in the nervous system and locomotor cilia of L. stagnalis, the observed distribution of isoforms was unanticipated. Specifically, neurons of other organisms have generally been reported to contain substantial amounts of both detyrosinated α-tubulin and acetylated α-tubulin. Our results indicate that such findings cannot be generalized across all species. L. stagnalis, with its well studied nervous system and unusual distribution of tubulin isoforms, may prove to be particularly useful for studying the roles of tubulin isoforms in microtubule function and cell activity.
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  • 31
    ISSN: 1432-0878
    Keywords: Circadian rhythm ; Colocalization ; Immunocytochemistry ; Brain (CNS), invertebrate ; Optic lobe ; Pigment-dispersing hormone, insect ; Leucophaea maderae (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Several lines of evidence suggest that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla are involved in the circadian system of insects. The present study provides a detailed analysis of the anatomical and neurochemical organization of the accessory medulla in the brain of the cockroach Leucophaea maderae. We show that the accessory medulla is compartmentalized into central dense nodular neuropil surrounded by a shell of coarse fibers. It is innervated by neurons immunoreactive to antisera against serotonin and the neuropeptides allatostatin 7, allatotropin, corazonin, gastrin/cholecystokinin, FMRFamide, leucokinin I, and pigment-dispersing hormone. Some of the immunostained neurons appear to be local neurons of the accessory medulla, whereas others connect this neuropil to various brain areas, including the lamina, the contralateral optic lobe, the posterior optic tubercles, and the superior protocerebrum. Double-label experiments show the colocalization of immunoreactivity against pigment-dispersing hormone with compounds related to FMRFamide, serotonin, and leucokinin I. The neuronal and neurochemical organization of the accessory medulla is consistent with the current hypothesis for a role of this brain area as a circadian pacemaking center in the insect brain.
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  • 32
    ISSN: 1432-0878
    Keywords: Neuropeptide Y ; Gastroenteropancreatic (GEP) endocrine system ; Development, ontogenetic ; Vitellointestinal duct ; Pancreas, exocrine ; Pancreas, endocrine ; Immunocytochemistry ; Scyliorhinus torazame (Elasmobranchii)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract This immunocytochemical study was carried out to elucidate the ontogenetic development of neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system of the cloudy dogfish, Scyliorhinus torazame. Immunostained cells first appeared in the pancreas of the embryo at the 15-mm stage, and were also detected in the vitellointestinal duct of the yolk stalk at the 20-mm stage. These cells were polymorphic, with occasional processes that were sometimes directed toward the vascular wall or into the cavity of the vitellointestinal duct. At the 34-mm stage, immunostained cells could also be found in the proximal part of the spiral intestine and, by the 74-mm stage, immunopositive cells were present in the gastric mucosa. In the gut and pancreas, the cells gradually increased in number with development, whereas in the vitellointestinal duct and internal yolk sac, they decreased and seemed to disappear following hatching. Thus, in juveniles, the distribution of the neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system had attained that of adults. Electron-microscopic immunocytochemistry demonstrated that, in the labeled cells of the vitellointestinal duct, the neuropeptide Y-like antigen was located in cytoplasmic granules, as in the cells of the gut and pancreas.
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  • 33
    ISSN: 1432-0878
    Keywords: Key words: Circadian rhythm ; Colocalization ; Immunocytochemistry ; Brain (CNS) ; invertebrate ; Optic lobe ; Pigment-dispersing hormone ; insect ; Leucophaea maderae (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Several lines of evidence suggest that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla are involved in the circadian system of insects. The present study provides a detailed analysis of the anatomical and neurochemical organization of the accessory medulla in the brain of the cockroach Leucophaea maderae. We show that the accessory medulla is compartmentalized into central dense nodular neuropil surrounded by a shell of coarse fibers. It is innervated by neurons immunoreactive to antisera against serotonin and the neuropeptides allatostatin 7, allatotropin, corazonin, gastrin/cholecystokinin, FMRFamide, leucokinin I, and pigment-dispersing hormone. Some of the immunostained neurons appear to be local neurons of the accessory medulla, whereas others connect this neuropil to various brain areas, including the lamina, the contralateral optic lobe, the posterior optic tubercles, and the superior protocerebrum. Double-label experiments show the colocalization of immunoreactivity against pigment-dispersing hormone with compounds related to FMRFamide, serotonin, and leucokinin I. The neuronal and neurochemical organization of the accessory medulla is consistent with the current hypothesis for a role of this brain area as a circadian pacemaking center in the insect brain.
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  • 34
    ISSN: 1432-0878
    Keywords: Key words Retina ; NOS ; Immunocytochemistry ; Synaptic connectivity ; Guinea pig
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Immunocytochemical methods with an antiserum against neuronal nitric oxide synthase (NOS) were applied to identify the morphology and synaptic connectivity of NOS-like immunoreactive neurons in the guinea pig retina. In the present study, two types of amacrine cells were labeled with anti-NOS antisera. Type 1 cells had large somata located in the inner nuclear layer (INL) with long, sparsely branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). The somata of type 2 cells (smaller diameters) were located in the INL. Some displaced amacrine cells in the ganglion cell layer were labeled. The soma size of the displaced amacrine cells was similar to that of the type 2 amacrine cells. However, processes originating from type 2 amacrine cells and displaced amacrine cells stratified mainly in strata 1 and 5, respectively. Some cone bipolar cells were weakly NOS-immunoreactive. The synaptic connectivity of NOS-like immunoreactive amacrine cells was identified in the IPL by electron microscopy. NOS-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in all strata of the IPL. The most frequent postsynaptic targets of NOS-immunoreactive amacrine cells were other amacrine cell processes. Cone bipolar cells were postsynaptic to NOS-labeled amacrine cells in all strata of the IPL. Labeled amacrine cells synapsing onto ganglion cells were found only in sublamina b. A few synaptic contacts were observed between labeled cell processes. In the outer plexiform layer, dendrites of labeled bipolar cells made basal contact with cone pedicles or formed a synaptic triad opposed to a synaptic ribbon of cone pedicles.
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  • 35
    ISSN: 1432-0878
    Keywords: Epididymis ; Efferent ducts ; Cell culture ; Immunocytochemistry ; Immunoprecipitation ; Man
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The way in which the human epididymis modifies spermatozoa during their sojourn in this structure might be clarified by knowledge of the nature of its secretions. We have examined the presence of several lysosomal hydrolases in human epididymal tissue and fluids, and their synthesis and secretion by monolayer cultures. Tissues were obtained from men undergoing orchidectomy for prostatic carcinoma. The enzymes cathepsin D and acid α-glucosidase were localised in the lysosomes of epithelial cells from the corpus epididymidis, by an immunocytochemical technique. Cathepsin D was also found in epithelial cells of the efferent ducts within lysosomes, apical vesicles and multivesicular bodies. No immunolocalisation of acid glucosidase in the efferent ducts or on the microvilli of the corpus was demonstrable. Cathepsin D, β-hexosaminidase (N-acetylglucosaminidase) and α-glucosidase were measurable in the luminal fluid from the human corpus epididymidis; β-hexosaminidase was secreted into the culture medium by confluent monolayers of epididymal and efferent duct cells. Immunoprecipitation of cell extracts and culture medium of these cultures incubated with 35S-methionine revealed that the precursors of cathepsin D and β-hexosaminidase were synthesized and secreted by such monolayers. Thus, active lytic enzymes are secreted by the human epididymis and could modify sperm membranes.
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  • 36
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    Cell & tissue research 280 (1995), S. 541-548 
    ISSN: 1432-0878
    Keywords: Musle, striated, skeletal ; Regeneration ; Myosin ; Immunocytochemistry ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Indirect immunofluorescence was used to localize embryonic myosin heavy chains in soleus, adductor longus, tibialis anterior, plantaris, and extensor digitorum longus muscles of 6-month-old rats. A monoclonal antibody (2B6), specifically recognizing rat embryonic myosin, was applied to unfixed, transverse, frozen sections. The number of embryonic myosin-positive (EMP) extrafusal fibers was expressed as a percentage of the total number of fibers. EMP extrafusal fibers were only seen in the soleus and adductor longus muscles, both postural muscles. Approximately 1% of the soleus muscle fibers appeared positively stained for embryonic myosin. The majority of such fibers had a small diameter (〈500 ν), appeared intensely fluorescent, and typically contained central nuclei. Re-expression of embryonic myosin due to spontaneous fiber denervation is not a likely factor in this study, since alpha-bungarotoxin and N-CAM localization were restricted to the motor end-plate region of EMP fibers. Since embryonic myosin was shown to disappear in all normal-sized myofibers by 2 to 3 months of age, the results suggest that the EMP extrafusal fibers seen in postural muscles of 6 to 12-month-old animals are regenerating myofibers. We speculate that a small number of muscle fibers may be regenerating in normal, adult postural muscles, in response to fiber damage possibly caused by excessive recruitment or overloading.
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  • 37
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    Cell & tissue research 280 (1995), S. 549-560 
    ISSN: 1432-0878
    Keywords: Enteric nervous system ; Immunocytochemistry ; Calretinin ; Calbindin ; Bombesin ; Small intestine ; Guinea-pig
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Light- and electron-microscopic studies were used to investigate connections between specific subgroups of neurons in the myenteric plexus of the guineapig small intestine. Inputs to two classes of calretinin-immunoreactive (IR) nerve cells, longitudinal muscle motor neurons and ascending interneurons, were examined. Inputs from calbindin-IR primary sensory neurons and from three classes of descending interneurons were studied. Electron-microscopic analysis showed that calbindin-IR axons formed two types of inputs, synapses and close contacts, on calretinin-IR neurons. About 40% of inputs to the longitudinal muscle motor neurons and 70% to ascending interneurons were calbindin-IR. Approximately 50% of longitudinal muscle motor neurons were surrounded by bombesin-IR dense pericellular baskets and 40% by closely apposed varicosities. At the electron-microscope level, the bombesin-IR varicosities were found to form synapses and close contacts with the motor neurons. Dense pericellular baskets with bombesin-IR surrounded 36% of all ascending interneurons, and a further 17% had closely apposed varicosities. Somatostatin-and 5-HT-IR descending interneurons provided no dense pericellular baskets to calretinin-IR nerve cells. Thus, calretinin-IR, longitudinal muscle motor neurons and ascending interneurons receive direct synaptic inputs from intrinsic primary sensory neurons and from non-cholinergic, bombesin-IR, descending interneurons.
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  • 38
    ISSN: 1432-0878
    Keywords: Laminin ; Nerve tracts ; Ontogenetic development ; Brain ; Immunocytochemistry ; Mouse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Laminin, a large glycoprotein of the basement membrane that promotes the growth of nerve cell processes in vitro has also been detected in the brains of developing embryos in situ where it is postulated to promote or guide neural outgrowth. We have investigated the histological and developmental patterns of a receptor to a specific pentapeptide sequence in the A chain of the laminin molecule (PA22-2 or IKVAV) that has been identified as a neuron growth-promoting sequence. Standard immunocytochemical procedures were used to localize the receptor by means of a polyclonal antibody to affinity-purified receptor (MR=110 kDa) from mouse brains. Results for postnatal stages (P) stages (P 1,7,8,25,30,and adult) show that the 110 kDa receptor is localized in fibers in the cortex and hippocampus, in astroglial cells at the surface of the cortex, and in neuronal cell bodies in the hippocampus. In contrast, the A-chain ligand is localized in cell bodies in the same regions at P stages. For embryonic stages (E) (E 14 and E 16) the receptor is localized in bundles of fibers in the superficial and deep cortical layers, and in cell bodies in these regions at E 14 only. Staining for the A chain ligand of the receptor was first seen postnatally. We speculate that the inverse histological pattern of receptor and ligand with respect to cell bodies and fibers may reflect a role in controlling axon guidance during development or repair during regeneration.
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  • 39
    ISSN: 1432-0878
    Keywords: Dopamine ; Serotonin ; Tyrosine hydroxylase ; Immunocytochemistry ; Nervous system, insect-Gryllus bimaculatus (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The cellular localization of the biogenic amines dopamine and serotonin was investigated in the ventral nerve cord of the cricket, Gryllus bimaculatus, using antisera raised against dopamine, β-tyrosine hydroxylase and serotonin. Dopamine-(n〈-70) and serotonin-immunoreactive (n〈-120) neurones showed a segmental arrangement in the ventral nerve cord. Some neuromeres, however, did not contain dopamine-immunoreactive cell bodies. The small number of stained cells allowed complete identification of brain and thoracic cells, including intersegmentally projecting axons and terminal arborizations. Dopamine-like immunostaining was found primarily in plurisegmental interneurones with axons descending to the soma-ipsilateral hemispheres of the thoracic and abdominal ganglia. In contrast, serotonin-immunostaining occurred predominantly in interneurones projecting via soma-contralaterally ascending axons to the thorax and brain. In addition, serotonin-immunoreactivity was also present in efferent cells and afferent elements. Serotonin-immunoreactive, but no dopamine-immunoreactive, varicose fibres were observed on the surface of some peripheral nerves. Varicose endings of both dopamine-and serotonin-immunoreactive neurones occurred in each neuromere and showed overlapping neuropilar projections in dorsal and medial regions of the thoracic ganglia. Ventral associative neuropiles lacked dopamine-like immunostaining but were innervated by serotonin-immunoreactive elements. A colocalization of the two amines was not observed. The topographic representation of neurone types immunoreactive for serotonin and dopamine is discussed with respect to possible modulatory functions of these biogenic amines in the central nervous system of the cricket.
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  • 40
    ISSN: 1432-0878
    Keywords: Cathepsin E ; Aspartic proteinase ; Osteoclasts ; Immunocytochemistry ; Rat (WKA)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The immunocytochemical localization of cathepsin E, a non-lysosomal aspartic proteinase, was investigated in rat osteoclasts using the monospecific antibody to this protein. At the light-microscopic level, the preferential immunoreactivity for cathepsin E was found at high levels in active osteoclasts in the physiological bone modeling process. Neighboring osteoblastic cells were devoid of its immunoreactivity. At the electron-microscopic level, cathepsin E was exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts and the eroded bone surface. Cathepsin E was also concentrated in some endocytotic vacuoles of various sizes in the vicinity of the ruffled border membrane, some of which appeared to be secondary lysosomes containing the phagocytosed materials. These results strongly suggest that this enzyme is involved both in the extracellular degradation of the bone organic matrix and in the intracellular breakdown of the ingested substances in osteoclasts.
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  • 41
    ISSN: 1432-0878
    Keywords: Key words: Laminin ; Nerve tracts ; Ontogenetic development ; Brain ; Immunocytochemistry ; Mouse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Laminin, a large glycoprotein of the basement membrane that promotes the growth of nerve cell processes in vitro has also been detected in the brains of developing embryos in situ where it is postulated to promote or guide neural outgrowth. We have investigated the histological and developmental patterns of a receptor to a specific pentapeptide sequence in the A chain of the laminin molecule (PA22-2 or IKVAV) that has been identified as a neuron growth-promoting sequence. Standard immunocytochemical procedures were used to localize the receptor by means of a polyclonal antibody to affinity-purified receptor (MR=110 kDa) from mouse brains. Results for postnatal stages (P) stages (P 1,7,8,25,30,and adult) show that the 110 kDa receptor is localized in fibers in the cortex and hippocampus, in astroglial cells at the surface of the cortex, and in neuronal cell bodies in the hippocampus. In contrast, the A-chain ligand is localized in cell bodies in the same regions at P stages. For embryonic stages (E) (E 14 and E 16) the receptor is localized in bundles of fibers in the superficial and deep cortical layers, and in cell bodies in these regions at E 14 only. Staining for the A chain ligand of the receptor was first seen postnatally. We speculate that the inverse histological pattern of receptor and ligand with respect to cell bodies and fibers may reflect a role in controlling axon guidance during development or repair during regeneration.
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  • 42
    ISSN: 1432-0878
    Keywords: Key words: Trans-differentiation ; Proliferation ; Bromodeoxyuridine ; Immunocytochemistry ; Regeneration ; Ciona intestinalis (Tunicata)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. In this study, we use three monoclonal antibodies that recognise antigens present in the central nervous system of the ascidian Ciona intestinalis to study regeneration and post-metamorphic development of the neural ganglion. We have also used bromodeoxyuridine labelling to study generation of the neuronal precursor cells. The first antibody, CiN 1, recognises all neurones in the ganglion, whereas the second, CiN 2, recognises only a subpopulation of the large cortical neurones. Western blotting studies show that CiN 2 recognises two membrane-bound glycoproteins of apparent Mr 129 and 100 kDa. CiN 1 is not reactive on Western blots. Immunocytochemical studies with these antibodies show that CiN 1-immunoreactive neurone-like cells are present at the site of regeneration as early as 5–7 days post-ablation, a sub-population of CiN 2-immunoreactive cells being detected by 9–12 days post-ablation. The third antibody, ECM 1, stains extracellular matrix components and recognises two diffuse bands on Western blots of whole-body and ganglion homogenates. The temporal and spatial pattern of appearance of CiN 1 and CiN 2 immunoreactivity both during post-metamorphic development and in regeneration occurs in the same sequence in both processes. Studies with bromodeoxyuridine show labelled nuclei in some neurones in the regenerating ganglion. Plausibly these originate from the dorsal strand, an epithelial tube that reforms by cell proliferation during the initial phases of regeneration. A second population of cells, the large cortical neurones, do not incorporate bromodeoxyuridine and thus must have been born prior to the onset of regeneration. This latter finding indicates a mechanism involving trans-differentiation of other cell types or differentiation of long-lived totipotent stem cells.
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  • 43
    ISSN: 1432-0878
    Keywords: Key words: Choline acetyltransferase ; Cholinergic neuron ; Visual system ; Bolwig’s organ ; Immunocytochemistry ; In situ hybridization ; Drosophila melanogaster (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Choline acetyltransferase (ChAT) is the enzyme catalyzing the biosynthesis of acetylcholine and is considered to be a phenotypically specific marker for cholinergic neurons. We have examined the distribution of ChAT-expressing neurons in the larval nervous system of Drosophila melanogaster by three different but complementary techniques: in situ hybridization with a cRNA probe to ChAT messenger RNA, immunocytochemistry using a monoclonal anti-ChAT antibody, and X-gal staining of transformed animals carrying a reporter gene composed of 7.4  kb of 5′ flanking DNA from the ChAT gene fused to a lacZ reporter gene. All three techniques demonstrated ChAT-expressing neurons in the larval visual system. In embryos, the photoreceptor organ (Bolwig’s organ) exhibited strong cRNA hybridization signals. The optic lobe of late third-instar larvae displayed ChAT immunoreactivity in Bolwig’s nerve and a neuron close to the insertion site of the optic stalk. This neuron’s axon ran in parallel with Bolwig’s nerve to the larval optic neuropil. This neuron is likely to be a first-order interneuron of the larval visual system. Expression of the lacZ reporter gene was also detected in Bolwig’s organ and the neuron stained by anti-ChAT antibody. Our observations indicate that acetylcholine may be a neurotransmitter in the larval photoreceptor cells as well as in a first-order interneuron in the larval visual system of Drosophila melanogaster.
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  • 44
    ISSN: 1432-0878
    Keywords: Pituitary ; Gonadotrops ; Crinophagy ; Electron microscopy ; Enzyme cytochemistry ; Immunocytochemistry ; Autoradiography ; Catfish, Clarias gariepinus (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The possible function of globules and irregular membrane-bound masses in the gonadotropin cells of the pituitary of Clarias gariepinus was studied. Strong secretory stimulation led to the disappearance of the secretory granules from gonadotropin cells but globules and irregular masses remained present. Acid phosphatase was detected enzyme-cytochemically in both globules and irregular masses. Radiolabelling with tritiated amino acids followed by autoradiography demonstrated that globules received radioactive material after secretory granules. The latter received radioactive material within 75 min of administration of radioactive amino acids but globules and irregular masses did not. Although some globules became radioactively labelled within 24 h of the administration of radioactive amino acids, irregular masses remained unlabelled during this period. Secretory granules reacted positively with antisera against α and β gonadotropin subunits, whereas globules and irregular masses only reacted with the antiserum against the β subunit. A moderate anti-7B2 immunoreactivity was demonstrated in secretory granules and globules, whereas irregular masses labelled strongly. The combined cytological results indicate that globules and irregular masses are degradative, possibly crinophagic structures which develop by fusional events from secretory granules to globules and then to irregular masses.
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  • 45
    ISSN: 1432-0878
    Keywords: Key words: Interleukin ; Stellate reticulum ; Immunocytochemistry ; Epidermal growth factor ; Interleukin-1 receptor type I messenger RNA ; Tooth eruption ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Immunolocalization of interleukin-1α in the first mandibular molars of rats from day 0–12 postnatally showed that the protein was localized in the epithelial stellate reticulum adjacent to the dental follicle. Staining of the stellate reticulum was most prominent in the early days postnatally and was absent by postnatal day 11. Injection of epidermal growth factor into rats at day 0 greatly increased the intensity of the staining for interleukin-1α in the stellate reticulum. Epidermal growth factor (EGF) enhanced the gene expression of interleukin-1α in stellate reticulum cells in vitro, and this study suggests there is enhanced translation of interleukin-1α messenger RNA in the stellate reticulum following EGF injection. In turn, the interleukin-1α may exert its effect on the dental follicle cells adjacent to the stellate reticulum because EGF also enhanced expression of the interleukin-1 receptor type I messenger RNA in cultured dental follicle cells as well as enhancing its expression in vivo. In view of the fact that injection of EGF will stimulate precocious eruption of teeth, its stimulus of interleukin-1α synthesis in the stellate reticulum may be the mechanism by which EGF initiates a cascade of molecular events to signal the onset of tooth eruption.
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  • 46
    ISSN: 1432-0878
    Keywords: Key words: Defence mechanisms ; Encapsulation ; Granulocytes ; Immunocytochemistry ; Parasitism ; Perkinsus sp. (Protozoa) ; Tapes semidecussatus (Mollusca
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Parasitosis by the trophozoite protozoan Perkinsus sp. (Apicomplexa, Perkinsea) induces in the gill filaments of the clam Tapes semidecussatus (Mollusca, Bivalvia) a cellular reaction, which is constituted by infiltrated granulocytes. This cellular reaction has characteristics of those of a holocrine gland, since the parasites are encapsulated by the secretion product of the granulocytes after cell death. An enriched fraction of prezoosporangia and their ass ociated capsule was obtained after culture of the parasitized gills in fluid thioglycollate medium. Specific polypeptides from this fraction were separated by SDS-PAGE and isolated for rabbit immunizations. The serum obtained against an Mr 225 kDa polypeptide, revealed its exclusive localization in the capsule and in the granules of the infiltrated granulocytes, thus indicating that this polypeptide is synthesized by these cells and secreted, in a polarized way, around the trophozoites resulting in t heir encapsulation. Selective deglycosylation of the polypeptide, by Endo H and alkaline β-elimination, did not show an effect on its molecular weight or antibody recognition. Furthermore, the absence of the 225 kDa band in the Western-blots of non-parasitized gills indicated the specific association of this polypeptide with the parasitosis. Finally, this is the first tissue-specific factor described in molluscs in relation to defence mechanisms.
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  • 47
    ISSN: 1432-0878
    Keywords: Key words: Pituitary ; Gonadotrops ; Crinophagy ; Electron microscopy ; Enzyme cytochemistry ; Immunocytochemistry ; Autoradiography ; Catfish ; Clarias gariepinus (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The possible function of globules and irregular membrane-bound masses in the gonadotropin cells of the pituitary of Clarias gariepinus was studied. Strong secretory stimulation led to the disappearance of the secretory granules from gonadotropin cells but globules and irregular masses remained present. Acid phosphatase was detected enzyme-cytochemically in both globules and irregular masses. Radiolabelling with tritiated amino acids followed by autoradiography demons trated that globules received radioactive material after secretory granules. The latter received radioactive material within 75 min of administration of radioactive amino acids but globules and irregular masses did not. Although some globules became radioactively labelled within 24 h of the administration of radioactive amino acids, irregular masses remained unlabelled during this period. Secretory granules reacted positively with antisera against α and β gonadotropin subunits, whereas globules and irregular masses only reacted with the antiserum against the β subunit. A moderate anti-7B2 immunoreactivity was demonstrated in secretory granules and globules, whereas irregular masses labelled strongly. The combined cytological results indicate that globules and irregular masses are degradative, possibly crinophagic structures which develop by fusional events from secretory granules to globules and then to irregular masses.
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  • 48
    ISSN: 1432-0878
    Keywords: Melanin-concentrating hormone ; Immunocytochemistry ; Development, ontogenetic ; Sparus auratus (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The development of the hypothalamic melanin-concentrating hormone (MCH) system of the teleost Sparus auratus has been studied by immunocytochemistry using an anti-salmon MCH serum. Immunoreactive perikarya and fibers are found in embryos, larvae, and juvenile specimens. In juveniles, most labeled neurons are present in the nucleus lateralis tuberis; some are dispersed in the nucleus recessus lateralis and nucleus periventricularis posterior. From the nucleus lateralis tuberis, MCH neurons project a conspicuous tract of fibers to the ventral hypothalamus; this penetrates the pituitary stalk and reaches the neurohypophysis. Most fibers end close to the cells of the pars intermedia, and some reach the adenohypophysial rostral pars distalis. Immunoreactive fibers can also be seen in extrahypophysial localizations, such as the preoptic region and the nucleus sacci vasculosi. In embryos, MCH-immunoreactive neurons first appear at 36 h post-fertilization in the ventrolateral margin of the developing hypothalamus. In larvae, at 4 days post-hatching, perikarya can be observed in the ventrolateral border of the hypothalamus and in the mid-hypothalamus, near the ventricle. At 26 days post-hatching, MCH perikarya are restricted to the nucleus lateralis tuberis. The neurohypophysis possesses MCH-immunoreactive fibers from the second day post-hatching. The results indicate that MCH plays a role in larval development with respect to skin melanophores and cells that secrete melanocyte-stimulating hormone.
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  • 49
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    Cell & tissue research 280 (1995), S. 123-131 
    ISSN: 1432-0878
    Keywords: Differentiation ; 5′-Nucleotidase ; Immunocytochemistry ; PC12 cells ; Synaptophysin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract 5′-Nucleotidase hydrolyzes 5′-mononucleotides to their nucleosides but is also thought to have a function in neuronal differentiation and synapse formation. The distribution of the enzyme, a glycosyl-phosphatidylinositol-anchored sialoglycoprotein, was investigated in PC12 cells using immunofluorescence microscopy. 5′-Nucleotidase was located both in intracellular compartments and at the cell surface. There was no principal difference in the cellular distribution between undifferentiated cells and after neuritogenic differentiation by nerve growth factor. Intracellularly, 5′-nucleotidase often revealed a sickle-shaped perinuclear distribution and a dotted pattern throughout the cytoplasm, including that of neurites and growth cones. The intracellular distribution was clearly different from that of the synaptic vesicle protein synaptophysin. However, the dotted fluorescence resembled that obtained after uptake of the endosomal marker acridine orange. 5′-Nucleotidase was present on the entire cell surface including all neurites formed after differentiation. There was no increase in 5′-nucleotidase fluorescence at synapse-like contacts between the tips of neurites and other PC12 cells. Surfacelocated 5′-nucleotidase could no longer be detected after the application of glycosyl-phosphatidylinositol-specific phospholipase C to cultured cells. This treatment did not affect PC12 cell differentiation. Our results thus reveal 5′-nucleotidase both at the surface and within organelles and suggest that PC12 cells may be used as a model system for the study of the physiological function of 5′-nucleotidase in neural cells.
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  • 50
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    Cell & tissue research 280 (1995), S. 123-131 
    ISSN: 1432-0878
    Keywords: Key words: Differentiation ; 5′-Nucleotidase ; Immunocytochemistry ; PC12 cells ; Synaptophysin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. 5′-Nucleotidase hydrolyzes 5′-mononucleotides to their nucleosides but is also thought to have a function in neuronal differentiation and synapse formation. The distribution of the enzyme, a glycosyl-phosphatidylinositol-anchored sialoglycoprotein, was investigated in PC12 cells using immunofluorescence microscopy. 5′-Nucleotidase was located both in intracellular compartments and at the cell surface. There was no principal difference in the cellular distrib ution between undifferentiated cells and after neuritogenic differentiation by nerve growth factor. Intracellularly, 5′-nucleotidase often revealed a sickle-shaped perinuclear distribution and a dotted pattern throughout the cytoplasm, including that of neurites and growth cones. The intracellular distribution was clearly different from that of the synaptic vesicle protein synaptophysin. However, the dotted fluorescence resembled that obtained after uptake of the endosomal marker acridine orange. 5′-Nucleotidase was present on the entire cell surface including all neurites formed after differentiation. There was no increase in 5′-nucleotidase fluorescence at synapse-like contacts between the tips of neurites and other PC12 cells. Surface-located 5′-nucleotidase could no longer be detected after the application of glycosyl-phosphatidylinositol-specific phospholipase C to cultured cells. This treatment did not affect PC12 cell differentiation. Our results thus reveal 5′ -nucleotidase both at the surface and within organelles and suggest that PC12 cells may be used as a model system for the study of the physiological function of 5′-nucleotidase in neural cells.
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  • 51
    ISSN: 1432-0878
    Keywords: Key words: Subcommissural organ ; Secretory glycoproteins ; Antibodies ; Immunochemistry ; Immunocytochemistry ; Dogfish ; Scyliorhinus canicula (Elasmobranchii)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The subcommissural organ of the dogfish, Scyliorhinus canicula (L), has been investigated by use of antibodies and lectins applied to blots and tissue sections processed for light and electron microscopy. Antibodies have been raised against each of the bands that have previously been identified in immunoblots by the use of antisera raised against secretory glycoproteins extracted from the dogfish subcommissural organ, viz., the 600-kDa band and two gel regions including the 475 to 400-kDa and the 145-kDa bands obtained from preparative gels; they are referred to as Ab-600, Ab-475/400, and Ab-145. These antisera and the lectins concanavalin A and wheat germ agglutinin have been used for the staining of: (1) blots of extracts of the dogfish subcommissural organ and optic tectum; (2) tissue sections of the dogfish brain. The findings indicate that the bands of 600, 475 and 400 kDa contain compounds that should be regarded as secretory glycoproteins of the dogfish subcommissural organ. The 600-kDa and 400-kDa bands are labeled by concanavalin A; wheat germ agglutinin labels the 475-kDa band strongly and the other two weakly. Ab-600 reacts with the bands at 600, 475 and 400 kDa and stains materials stored in the rough endoplasmic reticulum and secretory granules of 200–600 nm in diameter. The 600-kDa compound is probably a precursor form. Ab-475/400 stains the same three bands revealed by Ab-600; immunocytochemically, it reacts with two types of secretory granules (200–600 and 800–1200 nm in diameter) but it does not label the rough endoplasmic reticulum. Ab-145 reveals the bands at 600, 475 and 400 kDa and a diffuse zone in the region of 145 kDa; in light-microscopic immunocytochemistry, it behaves as Ab-475/400. The 475-kDa and 400-kDa glycoproteins, and a compound of approximately 145 kDa thus probably correspond to processed forms. Ab-475/400 stains granules present in cell processes ending on local blood vessels and at the leptomeninges. Since this antiserum selectively labels secretory granules, this finding may be taken as evidence for a basal route of secretion.
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  • 52
    ISSN: 1432-0878
    Keywords: Key words: Sauvagine ; Corticotropin-releasing factor ; Immunocytochemistry ; Interrenal gland ; Rana catesbeiana (Anura)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Immunocytochemistry was used to investigate the presence of corticotropin-releasing factor-like peptides in the interrenal (adrenal) glands of the bullfrog Rana catesbeiana by using specific antisera raised against synthetic nonconjugated rat/human corticotropin-releasing factor, urotensin I, and sauvagine. From these three antisera, covering a broad range of corticotropin-releasing factor-like immunoreactivities, only the sauvagine antiserum gave positive immunoreactivity. Sauvagine immunoreactivity was found in cortical cells grouped into cords in the renal zone of the interrenal gland. The central and subcapsular cords were less stained. Tyrosine hydroxylase-positive chromaffin cells were not sauvagine-immunoreactive. The immunoreactivity was abolished, in all cases, by previous immunoabsorption of the sauvagine antiserum with synthetic sauvagine (0.1 μM), but it was not eliminated by sucker (Catostomus commersoni) urotensin I, sole (Hippoglossoides elassodon) urotensin I, sucker corticotropin-releasing factor, rat/human corticotropin-releasing factor, or ovine corticotropin-releasing factor (0.1–10 μM). In a sauvagine radioimmunoassay, interrenal extracts displaced 125I-sauvagine from antiserum only partially, and not in parallel with the sauvagine standard curve. The results suggest that the sauvagine immunoreactivity in the R. catesbeiana interrenal gland may represent a novel sauvagine-like peptide.
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  • 53
    ISSN: 1432-0878
    Keywords: Key words: Pituitary hormones ; Immunocytochemistry ; Morphometry ; Metamorphosis ; Bufo arenarum (Anura)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The development and dynamics of thyrotropin (TSH), adrenocorticotropic hormone (ACTH), prolactin (PRL), and growth hormone (GH) cells have been studied using immunocytochemical techniques and rabbit antisera, raised against the relevant human hormone, in the pars distalis of Bufo arenarum larvae at different stages of development. The four types of cells studied were identified in different zones of the pars distalis: TSH cells occurred mainly in the centro-ventral zone, ACTH cells in the rostral and dorsal zones, GH cells in the central and caudal zones, and PRL cells in the anterior two-thirds of the gland. This distribution pattern does not show significant changes with development. Morphometry and stereology were used to evaluate the changes observed in the volume of the pars distalis and the immunoreactive cells during development. The former increased during larval growth and decreased throughout the metamorphic climax. The results obtained on cell number, volume density, and total volume suggest that, during larval growth (pre-prometamorphosis) of B. arenarum, TSH, PRL, GH and ACTH cells show a proliferative period with storage of their hormones; a second period involving hormone release occurs at the metamorphic climax.
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  • 54
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    Cell & tissue research 281 (1995), S. 367-374 
    ISSN: 1432-0878
    Keywords: Placenta ; Amniochorion ; Cytotrophoblast cells ; Immunoglobulin G ; Immunocytochemistry ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Maternal immunoglobulin-G (IgG) is known to be transported across the placental syncytiotrophoblast during the period when the human fetus is incapable of manufacturing these defensive molecules. In this study we investigated the possible role of the amniochorion, that surrounds the amniotic cavity in which the fetus lies, in the transfer of immunoglobulin. Endogenous IgG was localised in the amniochorion by confocal immunofluorescence microscopy and by ultrastructural labelling of ultrathin frozen tissue sections using the protein A-gold technique. Immunoreactivity was identified in the extracellular matrix tissues and necrotic amniotic epithelial cells. Healthy amniotic epithelial cells and cytotrophoblast cells of the chorion laeve were devoid o endogenous IgG. These results suggest a possible non-specific paracellular transport pathway between cytotrophoblast cells, which may conceivably contribute to the acquisition of passive immunity by the fetus, and offer a rational explanation for the presence of small quantities of maternal IgG in the amniotic fluid.
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  • 55
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    Cell & tissue research 281 (1995), S. 507-515 
    ISSN: 1432-0878
    Keywords: Key words: Microtubules ; Isoforms ; Nervous system ; Locomotion ; Cilia ; Immunocytochemistry ; Western blotting ; Lymnaea stagnalis (Mollusca)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Immunocytochemistry and Western blotting techniques demonstrated that the nervous system and foot of the pond snail Lymnaea stagnalis are rich sources of tubulin, which can be extracted and assembled in vi- tro in the presence of taxol. Various broad-spectrum antibodies raised against α-tubulin and β-tubulin yielded qualitatively similar results. One monoclonal antibody to trypanosome α−tubulin, however, labelled α-tubulin more strongly on both probed sections and Western blots. Cytochemistry and immunoblotting revealed that tyrosinated tubulin constitutes a large proportion of total α-tubulin in locomotor cilia of the foot and in axons of the nervous system. Detyrosinated tubulin also appeared to be abundant in the foot cilia but only a very faint band of detyrosinated tubulin was found on protein blots extracted from the central ganglia, and staining was barely detectable in central ganglia or peripheral nerves. Similarly, acetylated tubulin appeared to be abundant in foot cilia, but Western blotting indicated only low levels of acetylated tubulin in the nervous system. Immunocytochemistry indicated that, while most neurons possessed little or no acetylated tubulin, a small number of axons contained significant amounts of this isoform. Thus, while a large amount of tubulin was expected in the nervous system and locomotor cilia of L. stagnalis, the observed distribution of isoforms was unanticipated. Specifically, neurons of other organisms have generally been reported to contain substantial amounts of both detyrosinated α-tubulin and acetylated α-tubulin. Our results indicate that such findings cannot be generalized across all species. L. stagnalis, with its well studied nervous system and unusual distribution of tubulin isoforms, may prove to be particularly useful for studying the roles of tubulin isoforms in microtubule function and cell activity.
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  • 56
    ISSN: 1432-0878
    Keywords: Key words Coagulating gland ; Apocrine secretion ; Merocrine secretion ; Immunocytochemistry ; Immunoelectron microscopy ; Scanning electron microscopy ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The coagulating gland of the rat synthesizes two prevalent secretory proteins (transglutaminase and 115 K) that are discharched in a different manner, one being secreted in an apocrine fashion (transglutaminase) and the other one in a merocrine way (115 K). Differences in the intra- cellular pathway and the release of either protein were studied using immunofluorescence on semithin sections, immunoelectron microscopy of preembedding-processed chopper sections and postembedding-processed ultrathin sections of rat coagulating gland. Immunohistochemical staining using an anti-transglutaminase antibody resulted in dense labeling of the cytoplasm of secretory cells and their apical blebs, whereas the cisternae of the rough endoplasmic reticulum and the Golgi apparatus were completely unlabeled. When, on the contrary, the anti-115 K antiserum was used, dense labeling of the cisternae of the rough endoplasmic reticulum, the Golgi apparatus, and the secretory granules was seen. Intraluminal secretion was also labeled, but the secretory blebs remained unlabeled. Our findings show that, in the coagulating gland of the male rat, the two secretory proteins studied are processed in parallel, but at completely different intracellular pathways. They are released via different extrusion mechanisms. Transglutaminase is synthesized outside the endoplasmic reticulum, reaches the apical cell pole by free flow in the cytoplasm, and is released via apocrine blebs, the membranes of which appear to be derived from the apical plasma membrane. The protein 115 K, on the other hand, follows the classic route, being synthesized within the cisternae of rough endoplasmic reticulum, subsequently glycosylated in the Golgi apparatus, and released in a merocrine fashion. The mutual exclusion of the two secretory pathways and the regulation of the alternative release mechanism are still unresolved issues.
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  • 57
    ISSN: 1432-0878
    Keywords: Key words Cerebrovascular development and injury ; Hemangioma ; Angiogenesis ; Immunocytochemistry ; Adhesion molecules ; Conventional transmission and high-voltage electron microscopy ; Mouse (C57BL ; SJL/J) ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Blood vessels from the vasculature of mouse brains during postnatal development and from human brain tumors (hemangiomas) removed at biopsy were examined immunocytochemically by transmission electron microscopy (TEM) or high-voltage transmission electron microscopy (HVEM) to determine the expression of intercellular adhesion molecule-1 (ICAM-1). In the mouse brains, ICAM-1 was shown to be initially expressed on the luminal and abluminal endothelial cell (EC) surfaces on day 3 after birth. ICAM-1 intensity increased on the luminal EC surfaces and labeled vesiculotubular profiles (VTS, defined in the present report) between days 5 and 7. After 2 weeks and at 6 months after birth, ICAM-1 labeling was weak or absent on the luminal EC surfaces. The hemangiomas presented a strong ICAM-1 reaction product on the luminal EC surfaces of small and large blood vessels associated with the VTS, with a weaker labeling of the abluminal or adventitial aspects of larger blood vessels. TEM of vesiculovacuolar structures (VVOs) within ECs from arteries and veins also demonstrated reaction product for ICAM-1 labeling. Three-dimensional stereo-pair images in the HVEM enhanced the visualization of gold particles that were attached to the inner-delimiting membrane surfaces of EC VTS, and VVOs, respectively. These observations raise the possibility that the neonatal leukocytes and tumor cells may utilize these endothelial structures as a route across the developing and injured blood-brain barrier (BBB).
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  • 58
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    Cell & tissue research 295 (1999), S. 159-170 
    ISSN: 1432-0878
    Keywords: Key words Neuropeptides ; Perisympathetic organ ; Myotropin ; Visceral muscles ; Immunocytochemistry ; Insect nervous system ; Periplaneta americana (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A highly specific polyclonal antiserum has been raised against periviscerokinin, the first neuropeptide isolated from the perisympathetic organs of insects (Predel et al. 1995). In this study, two different neuronal systems with periviscerokinin-like immunoreactivity were distinguished in the central nervous system of the American cockroach: (1) An intrinsic neuronal network, restricted to the head-thoracic region, was formed by intersegmental projecting neurons of the brain, suboesophageal ganglion and metathoracic ganglion. In addition, groups of local interneurons occurred in the proto- and tritocerebrum. (2) A typical neurohormonal system was stained exclusively in the abdomen; it was represented by abdominal perisympathetic organs which were supplied by three cell clusters located in each unfused abdominal ganglion. As revealed by nickel backfills, most neurons with axons entering the perisympathetic organs contained a periviscerokinin-like peptide. Immunoreactive fibres left the perisympathetic organs peripherally, innervated the hyperneural muscle and ran via the link nerves/segmental nerves to the heart and segmental vessels. All visceral muscles innervated by periviscerokinin-immunoreactive fibres were shown to be sensitive to periviscerokinin, whereas the hindgut gave no specific response to this peptide.
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  • 59
    ISSN: 1432-0878
    Keywords: Key words: Nervous system ; insect ; DUM neuron ; Synapses ; Immunocytochemistry ; GABA ; Glutamate ; Locusta migratoria (Insecta) ; Schistocerca gregaria (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Dorsal unpaired median (DUM) neurones in the abdominal ganglia of the locust were impaled with microelectrodes and some were injected intracellularly with horseradish peroxidase so that their synapses could be identified in the electron microscope. Simultaneous recordings from DUM neurones in different abdominal ganglia revealed that they received common postsynaptic potentials from descending interneurones. Post-embedding immunocytochemistry using antibodies against GABA and glutamate was carried out on ganglia containing HRP-stained neurones. GABA-like immunoreactivity was found in 39% (n=82) of processes presynaptic to abdominal DUM neurones and glutamate-like immunoreactivity in 21% (n=42) of presynaptic processes. Output synapses from the DUM neurites were rarely observed within the neuropile. Structures resembling presynaptic dense bars but not associated with synaptic vesicles, were seen in some large diameter neurites.
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  • 60
    ISSN: 1432-0878
    Keywords: Key words: Leu-callatostatin ; Allatostatins ; Neuropeptides ; In situ hybridisation ; Immunocytochemistry ; Hindgut innervation ; Midgut endocrine cells ; Calliphora vomitoria ; Lucilia cuprina (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.
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  • 61
    ISSN: 1432-0878
    Keywords: Key words: Oocyte ; Yolk granules ; Ribonuclease ; Immunocytochemistry ; Bullfrog ; Rana catesbeiana (Anura)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.
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  • 62
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    Cell & tissue research 280 (1995), S. 549-560 
    ISSN: 1432-0878
    Keywords: Key words: Enteric nervous system ; Immunocytochemistry ; Calretinin ; Calbindin ; Bombesin ; Small intestine ; Guinea-pig
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Light- and electron-microscopic studies were used to investigate connections between specific subgroups of neurons in the myenteric plexus of the guinea-pig small intestine. Inputs to two classes of calretinin-immunoreactive (IR) nerve cells, longitudinal muscle motor neurons and ascending interneurons, were examined. Inputs from calbindin-IR primary sensory neurons and from three classes of descending interneurons were studied. Electron-microscopic analysis showed that calbindin-IR axons formed two types of inputs, synapses and close contacts, on calretinin-IR neurons. About 40% of inputs to the longitudinal muscle motor neurons and 70% to ascending interneurons were calbindin-IR. Approximately 50% of longitudinal muscle motor neurons were surrounded by bombesin-IR dense pericellular baskets and 40% by closely apposed varicosities. At the electron-microscope level, the bombesin-IR varicosities were found to form synapses and close contacts with the motor neurons. Dense pericellular baskets with bombesin-IR surrounded 36% of all ascending interneurons, and a further 17% had closely apposed varicosities. Somatostatin- and 5-HT-IR descending interneurons provided no dense pericellular baskets to calretinin-IR nerve cells. Thus, calretinin-IR, longitudinal muscle motor neurons and ascending interneurons receive direct synaptic inputs from intrinsic primary sensory neurons and from non-cholinergic, bombesin-IR, descending interneurons.
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  • 63
    ISSN: 1432-0878
    Keywords: Key words: Nitric oxide synthase ; Coronary vasculature ; Electron microscopy ; Immunocytochemistry ; Rat (Sprague Dawley) ; Guinea-pig (Dunkin Hartley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Ultrastructural investigation of nitric oxide synthase-immunoreactive nerves closely associated with blood vessels in rat and guinea-pig hearts revealed many labelled nerve fibres in the walls of the main branches of the coronary arteries, and in arterioles, capillaries and post-capillary venules. The number of nitric oxide synthase-containing nerve fibres associated with different vessels, even those of the same calibre, varied. Terminal regions of nitric oxide synthase-immunoreactive fibres were observed in the endocardium and myocardium. Nitric oxide synthase-labelled fibres displayed electron-dense immunoproduct in both varicose and intervaricose regions. Immunoreactive axonal varicosities contained both small and large synaptic vesicles. The characteristics of the nitric oxide synthase-immunoreactive nerve fibres observed in the heart and the possibility that these fibres represent the processes of intracardiac neurones and/or sensory neurones of extrinsic origin are discussed.
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  • 64
    ISSN: 1432-0878
    Keywords: Key words: Dopamine ; Serotonin ; Tyrosine hydroxylase ; Immunocytochemistry ; Nervous system ; insect ; Gryllus bimaculatus (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The cellular localization of the biogenic amines dopamine and serotonin was investigated in the ventral nerve cord of the cricket, Gryllus bimaculatus, using antisera raised against dopamine, β-tyrosine hydroxylase and serotonin. Dopamine- (n≤70) and serotonin-immunoreactive (n≤120) neurones showed a segmental arrangement in the ventral nerve cord. Some neuromeres, however, did not contain dopamine-immunoreactive cell bodies. The small number of stained cells allowed complete identification of brain and thoracic cells, including intersegmentally projecting axons and terminal arborizations. Dopamine-like immunostaining was found primarily in plurisegmental interneurones with axons descending to the soma-ipsilateral hemispheres of the thoracic and abdominal ganglia. In contrast, serotonin-immunostaining occurred predominantly in interneurones projecting via soma-contralaterally ascending axons to the thorax and brain. In addition, serotonin-immunoreactivity was also present in efferent cells and afferent elements. Serotonin-immunoreactive, but no dopamine-immunoreactive, varicose fibres were observed on the surface of some peripheral nerves. Varicose endings of both dopamine- and serotonin-immunoreactive neurones occurred in each neuromere and showed overlapping neuropilar projections in dorsal and medial regions of the thoracic ganglia. Ventral associative neuropiles lacked dopamine-like immunostaining but were innervated by serotonin-immunoreactive elements. A colocalization of the two amines was not observed. The topographic representation of neurone types immunoreactive for serotonin and dopamine is discussed with respect to possible modulatory functions of these biogenic amines in the central nervous system of the cricket.
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  • 65
    ISSN: 1432-0878
    Keywords: Key words: Neurohemal areas ; Neuropeptides ; Monoamines ; Immunocytochemistry ; Nervous system ; insect ; Gryllus bimaculatus (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The morphology and position of putative neurohemal areas in the peripheral nervous system (ventral nerve cord and retrocerebral complex) of the cricket Gryllus bimaculatus are described. By using antisera to the amines dopamine, histamine, octopamine, and serotonin, and the neuropeptides crustacean cardioactive peptide, FMRFamide, leucokinin 1, and proctolin, an extensive system of varicose fibers has been detected throughout the nerves of all neuromeres, except for nerve 2 of the prothoracic ganglion. Immunoreactive varicose fibers occur mainly in a superficial position at the neurilemma, indicating neurosecretory storage and release of neuroactive compounds. The varicose fibers are projections from central or peripheral neurons that may extend over more than one segment. The peripheral fiber varicosities show segment-specific arrangements for each of the substances investigated. Immunoreactivity to histamine and octopamine is mainly found in the nerves of abdominal segments, whereas serotonin im-munoreactivity is concentrated in subesophageal and terminal ganglion nerves. Immunoreactivity to FMRFamide and crustacean cardioactive peptide is widespread throughout all segments. Structures immunoreactive to leucokinin 1 are present in abdominal nerves, and proctolin immunostaining is found in the terminal ganglion and thoracic nerves. Codistribution of peripheral varicose fiber plexuses is regularly seen for amines and peptides, whereas the colocalization of substances in neurons has not been detected for any of the neuroactive compounds investigated. The varicose fiber system is regarded as complementary to the classical neurohemal organs.
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  • 66
    ISSN: 1432-0878
    Keywords: Key words: Compound eye ; Photoreceptor cells ; Ion pumps ; Polarity ; Immunocytochemistry ; Manduca sexta (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Immunohistochemical and physiological studies on various insect photoreceptors have demonstrated that the Na,K-ATPase (sodium pump) is restricted to the nonreceptive nonmicrovillar area of the plasma membrane. Here, we examined the distribution of the Na,K-ATPase in photoreceptor cells of the superposition-type compound eye in the moth Manduca sexta. Using immunofluorescent and immunogold cytochemistry, we show that the Na,K-ATPase is localized to both the nonmicrovillar and the microvillar parts of the plasma membrane. Manduca photoreceptors thus deviate from the common concept that the sodium pump and the molecular components of the photoreceptive machinery reside on different domains of the plasma membrane.
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  • 67
    ISSN: 1432-0878
    Keywords: Key words: Blood platelets ; Immunocytochemistry ; Electron microscopy ; Coated vesicles ; Clathrin ; Adhesive proteins ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Coated membranes and vesicles play an important role in receptor-mediated endocytosis and intracellular trafficking in various cell types, and are also present in blood platelets. Platelets take up certain proteins from the blood plasma, such as von Willebrand factor and fibrinogen, and these substances are transferred to storage granules. The receptors for these plasma proteins on the platelet plasma membrane have been well characterized, but morphological evidence for their transport to the storage granules is not yet available. In an attempt to clarify this aspect, we employed postembedding immunocytochemistry on platelets embedded in the acrylic resin LR White. Clathrin as the major coat component of coated vesicles was localized in the cytoplasm, on the plasmic faces of α-granules and the open canalicular system, and on the plasmic face of the plasma membrane. Colocalizations of the adhesive proteins, von Willebrand factor, fibrinogen and fibronectin, with clathrin could be observed at the same typical locations as coated vesicles were seen in Araldite-embedded material. These colocalizations have not been reported to date and furnish further evidence for a coated vesicle-mediated transport of blood plasma-derived adhesive proteins from their receptors on the outer plasma membrane to the α-granules.
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  • 68
    ISSN: 1432-0878
    Keywords: Blood platelets ; Immunocytochemistry ; Electron microscopy ; Coated vesicles ; Clathrin ; Adhesive proteins ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Coated membranes and vesicles play an important role in receptor-mediated endocytosis and intracellular trafficking in various cell types, and are also present in blood platelets. Platelets take up certain proteins from the blood plasma, such as von Willebrand factor and fibrinogen, and these substances are transferred to storage granules. The receptors for these plasma proteins on the platelet plasma membrane have been well characterized, but morphological evidence for their transport to the storage granules is not yet available. In an attempt to clarify this aspect, we employed postembedding immunocytochemistry on platelets embedded in the acrylic resin LR White. Clathrin as the major coat component of coated vesicles was localized in the cytoplasm, on the plasmic faces of α-granules and the open canalicular system, and on the plasmic face of the plasma membrane. Colocalizations of the adhesive proteins, von Willebrand factor, fibrinogen and fibronectin, with clathrin could be observed at the same typical locations as coated vesicles were seen in Araldite-embedded material. These colocalizations have not been reported to date and furnish further evidence for a coated vesicle-mediated transport of blood plasma-derived adhesive proteins from their receptors on the outer plasma membrane to the α-granules.
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  • 69
    ISSN: 1432-0878
    Keywords: Choline acetyltransferase ; Cholinergic neuron ; Visual system ; Bolwig's organ ; Immunocytochemistry ; In situ hybridization ; Drosophila melanogaster (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Choline acetyltransferease (ChAT) is the enzyme catalyzing the biosynthesis of acetylcholine and is considered to be a phenotypically specific marker for cholinergic neurons. We have examined the distribution of ChAT-expressing neurons in the larval nervous system of Drosophila melanogaster by three different but complementary techniques: in situ hybridization with a cRNA probe to ChAT messenger RNA, immunocytochemistry using a monoclonal anti-ChAT antibody, and X-gal staining of transformed animals carrying a reporter gene composed of 7.4 kb of 5′ flanking DNA from the ChAT gene fused to a lacZ reporter gene. All three techniques demonstrated ChAT-expressing neurons in the larval visual system. In embryos, the photoreceptor organ (Bolwig's organ) exhibited strong cRNA hybridization signals. The optic lobe of late third-instar larvae displayed ChAT immunoreactivity in Bolwig's nerve and a neuron close to the insertion site of the optic stalk. This neuron's axon ran in parallel with Bolwig's nerve to the larval optic neuropil. This neuron is likely to be a first-order interneuron of the larval visual system. Expression of the lacZ reporter gene was also detected in Bolwig's organ and the neuron stained by anti-ChAT antibody. Our observations indicate that acetylcholine may be a neurotransmitter in the larval photoreceptor cells as well as in a first-order interneuron in the larval visual system of Drosophila melanogaster.
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  • 70
    ISSN: 1432-0878
    Keywords: Vacuolar ATPase ; Proton pump ; Electrogenic potassium transport ; Marpighian tubules ; Immunocytochemistry ; Formica polyctena (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The presence of a vacuolar-type ATPase in Malpighian tubules of the ant Formica polyctena was investigated immunocytochemically, using antibodies to vacuolar ATPases of Manduca sexta midgut and bovine kidney. Specific labelling was observed at the brush border of the epithelium extending along the entire length of the tubules. These findings agree with the current view that a vacuolar ATPase is situated at the apical membrane of Malpighian tubule cells and other insect epithelial cells, being the energizing element of an electrogenic potassium pump. When antibodies were tested on tubules in different secretion conditions prior to fixation, no differences were observed in the distribution of the vacuolar ATPase.
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  • 71
    ISSN: 1432-0878
    Keywords: Key words: Vacuolar ATPase ; Proton pump ; Electrogenic potassium transport ; Malpighian tubules ; Immunocytochemistry ; Formica polyctena (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The presence of a vacuolar-type ATPase in Malpighian tubules of the ant Formica polyctena was investigated immunocytochemically, using antibodies to vacuolar ATPases of Manduca sexta midgut and bovine kidney. Specific labelling was observed at the brush border of the epithelium, extending along the entire length of the tubules. These findings agree with the current view that a vacuolar ATPase is situated at the apical membrane of Malpighian tubule cells and other insect epithelial cells, being the energizing element of an electrogenic potassium pump. When antibodies were tested on tubules in different secretion conditions prior to fixation, no differences were observed in the distribution of the vacuolar ATPase.
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  • 72
    ISSN: 1432-0878
    Keywords: Nervous system, insect ; DUM neuron ; Synapses ; Immunocytochemistry ; GABA ; Glutamate ; Locusta migratoria (Insecta) ; Schistocerca gregaria (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Dorsal unpaired median (DUM) neurones in the abdominal ganglia of the locust were impaled with microelectrodes and some were injected intracellularly with horseradish peroxidase so that their synapses could be identified in the electron microscope. Simultaneous recordings from DUM neurones in different abdominal ganglia revealed that they received common postsynaptic potentials from descending interneurones. Post-embedding immunocytochemistry using antibodies against GABA and glutamate was carried out on ganglia containing HRP-stained neurones. GABA-like immunoreactivity was found in 39% (n=82) of processes presynaptic to abdominal DUM neurones and glutamate-like immunoreactivity in 21% (n=42) of presynaptic processes. Output synapses from the DUM neurites were rarely observed within the neuropile. Structures resembling presynaptic dense bars but not associated with synaptic vesicles, were seen in some large diameter neurites.
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  • 73
    ISSN: 1432-0878
    Keywords: Immunocytochemistry ; Catch-relaxing peptide (CARP) ; Nervous system, central ; Nervous system, peripheral ; Helix pomatia (Mollusca)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Immunocytochemistry was performed on the nervous system of Helix by the use of an antibody raised against a myotropic neuropeptide, the catch-relaxing peptide (CARP), isolated from Mytilus edulis. In each ganglion of the central nervous system of Helix pomatia, numerous CARP-immunoreactive cell bodies and a dense immunoreactive fiber system could be observed with a dominancy in the cerebral and pedal ganglia. The majority of the immunoreactive neurons are unipolar, although multipolar neurons also occur. In the neuropil areas, CARP-immunoreactive fibers show extensive arborization, which may indicate a central role of CARP. CARP-immunoreactive elements could be observed in each investigated peripheral nerve and peripheral areas, namely in the intestine, heart, aorta, buccal mass, lips, and foot. However, CARP-immunoreactive cell bodies could only be demonstrated in the intestine and the foot musculature. Thin varicose CARP-immunoreactive fibers were observed over both muscle and gland cells in the different peripheral organs, suggesting a peripheral role of CARP. In vivo CARP injection into the body cavity (10-3, 10-4, 10-5 M) altered the general behavioral state of the animals and induced the relaxation of the musculature of the whole body wall indicating that CARP has a significant role in the regulation of muscle contraction.
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  • 74
    ISSN: 1432-0878
    Keywords: Leu-callatostatin ; Allatostatins ; Neuropeptides ; In situ hybridisation ; Immunocytochemistry ; Hindgut innervation ; Midgut endocrine cells ; Calliphora vomitoria, Lucilia cuprina (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.
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  • 75
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    Cell & tissue research 281 (1995), S. 569-572 
    ISSN: 1432-0878
    Keywords: Corticotropin-releasing factor ; Immunocytochemistry ; Gilthead sea bream ; Sparus aurata (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The distribution of perikarya and fibers containing corticotropin-releasing factor (CRF) was studied in the brain of the teleost Sparus aurata by immunocytochemistry using the peroxidase-antiperoxidase method. Antisera against rat CRF, arginine vasotocin, and human adrenocorticotropin (ACTH) were used. Most CRF-immunoreactive neurons were located in the nucleus lateralis tuberis, but they were absent from the nucleus preopticus, which only contained arginine vasotocin neurons. Few CRF perikarya were identified in the nucleus preopticus periventricularis and in the mesencephalic tegmentum. A conspicuous bundle of immunoreactive fibers ran along the diencephalic floor and pituitary stalk to end near the cells of the hypophysial pars intermedia. No CRF was seen near the adenohypophysial rostral pars distalis. Our results suggest that, in Sparus aurata, CRF is a releasing factor for melanotropic cells. Its role as a releasing factor for ACTH is discussed.
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  • 76
    ISSN: 1432-0878
    Keywords: Gap junction ; Intercellular junction ; Insect ; Arthropod ; Immunocytochemistry ; Hellothis virescens (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Gap junction-enriched fractions were prepared from larvae of the tobacco budworm Heliothis virescens using the NaOH procedure in the presence or absence of protease inhibitors and were analyzed by SDS-PAGE, immunoblotting and EM immunocytochemistry. Protease inhibitor fractions contained a 48-kDa protein in addition to the ∼10 proteins in fractions with and without inhibitors. Three polyclonal antibodies were used as probes for gap junction plaques and proteins: R16, against an ∼40-kDa candidate gap junction protein from Drosophila melanogaster; R17, against the 40-kDa candidate gap junction protein from H. virescens; and R18AP, an affinity purified antibody against a consensus sequence of N-terminal amino acids 2–21 of the H. virescens 40-kDa protein. R16, R17, and R18AP stain the 40- and 48-kDa proteins, R16 and R18AP stain a 64-kDa protein, and R16 stains an ∼30-kDa protein in the absence of inhibitors. Inclusion of protease inhibitors had no effect on gap junction ultrastructure. R16 and R17 label gap junction plaques in crude membrane and NaOH fractions, whereas R18AP exhibits only a low level of reactivity with gap junctions in crude membrane fractions and none with gap junctions in NaOH fractions. The results show that the 30-, 40-, 48- and 64-kDa proteins are immunologically related and are associated with gap junctions in H. virescens, the N-terminus of the 40-kDa protein is relatively inaccessible or easily lost, and the 48-kDa protein is protease-sensitive.
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  • 77
    ISSN: 1432-0878
    Keywords: Key words: Cathepsin E ; Aspartic proteinase ; Osteoclasts ; Immunocytochemistry ; Rat (WKA)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The immunocytochemical localization of cathepsin E, a non-lysosomal aspartic proteinase, was investigated in rat osteoclasts using the monospecific antibody to this protein. At the light-microscopic level, the preferential immunoreactivity for cathepsin E was found at high levels in active osteoclasts in the physiological bone modeling process. Neighboring osteoblastic cells were devoid of its immunoreactivity. At the electron-microscopic level, cathepsin E was exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts and the eroded bone surface. Cathepsin E was also concentrated in some endocytotic vacuoles of various sizes in the vicinity of the ruffled border membrane, some of which appeared to be secondary lysosomes containing the phagocytosed materials. These results strongly suggest that this enzyme is involved both in the extracellular degradation of the bone organic matrix and in the intracellular breakdown of the ingested substances in osteoclasts.
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  • 78
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    Cell & tissue research 281 (1995), S. 101-108 
    ISSN: 1432-0878
    Keywords: Key words: Somite ; Integrin ; Extracellular matrix ; structures ; Embryo ; Laminin ; Immunocytochemistry ; Macaca fascicularis (Primates)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The distribution of integrin subunits α6 and β1, and the α6β1 integrin ligand, laminin, was examined during somitogenesis in developmental stages 11, 13, and 16 in the long-tailed macaque, using peroxidase immunocytochemistry. Within differentiating somites in stage 11, α6 expression was observed in the sclerotome, basal surface of dermamyotomal cells adjacent to the basal lamina and on scattered cells throughout the dermamyotome. In further advanced somites in stages 13 and 16, α6 immunoreactivity became restricted to the myotome. α6 was expressed on mesenchymal core cells within the myocele of undifferentiated epithelioid somites and the ventromedial wall of somites commencing differentiation at each stage. β1 distribution resembled that of α6 in stage 11 somitic tissue, however, it remained present on myotome and sclerotome cells in the later stages, and was also expressed on dermatomal cells in stage 16. Laminin immunoreactivity, while more intense and prevalent than α6 and β1 in each stage examined, occurred on the same somite cell populations as the 2 integrin subunits. These results show a defined distribution of α6 on somitic tissue, and suggest this integrin is involved in somite differentiation. They also support a possible role for α6 in myoblast formation and migration. Overlapping of β1 and laminin immunoreactivity with that of α6 further suggests that α6 pairs with β1 as a functional heterodimer for laminin in defined somitic regions.
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  • 79
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    Cell & tissue research 281 (1995), S. 367-374 
    ISSN: 1432-0878
    Keywords: Key words: Placenta ; Amniochorion ; Cytotrophoblast cells ; Immunoglobulin-G ; Immunocytochemistry ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Maternal immunoglobulin-G (IgG) is known to be transported across the placental syncytiotrophoblast during the period when the human fetus is incapable of manufacturing these defensive molecules. In this study we investigated the possible role of the amniochorion, that surrounds the amniotic cavity in which the fetus lies, in the transfer of immunoglobulin. Endogenous IgG was localised in the amniochorion by confocal immunofluorescence microscopy and by ultrastructural labelling of ultrathin frozen tissue sections using the protein A-gold technique. Immunoreactivity was identified in the extracellular matrix tissues and necrotic amniotic epithelial cells. Healthy amniotic epithelial cells and cytotrophoblast cells of the chorion laeve were devoid of endogenous IgG. These results suggest a possible non-specific paracellular transport pathway between cytotrophoblast cells, which may conceivably contribute to the acquisition of passive immunity by the fetus, and offer a rational explanation for the presence of small quantities of maternal IgG in the amniotic fluid.
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  • 80
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    Cell & tissue research 281 (1995), S. 569-572 
    ISSN: 1432-0878
    Keywords: Key words: Corticotropin-releasing factor ; Immunocytochemistry ; Gilthead sea bream ; Sparus aurata (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The distribution of perikarya and fibers containing corticotropin-releasing factor (CRF) was studied in the brain of the teleost Sparus aurata by immunocytochemistry using the peroxidase-antiperoxidase method. Antisera against rat CRF, arginine vasotocin, and human adrenocorticotropin (ACTH) were used. Most CRF-immunoreactive neurons were located in the nucleus lateralis tuberis, but they were absent from the nucleus preopticus, which only contained arginine vasotocin neurons. Few CRF perikarya were identified in the nucleus preopticus periventricularis and in the mesencephalic tegmentum. A conspicuous bundle of immunoreactive fibers ran along the diencephalic floor and pituitary stalk to end near the cells of the hypophysial pars intermedia. No CRF was seen near the adenohypophysial rostral pars distalis. Our results suggest that, in Sparus aurata, CRF is a releasing factor for melanotropic cells. Its role as a releasing factor for ACTH is discussed.
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  • 81
    ISSN: 1432-0878
    Keywords: Key words Pigment-dispersing hormone ; Immunocytochemistry ; Central nervous system ; Gastropoda ; Helix pomatia ; Lymnaea stagnalis (Mollusca)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract By using an antiserum raised against a crustacean β-pigment-dispersing hormone (PDH), the distribution and chemical neuroanatomy of PDH-like immunoreactive neurons was investigated in the central nervous system of the gastropod snails, Helix pomatia and Lymnaea stagnalis. The number of immunoreactive cells in the Helix central nervous system was found to be large (700–900), whereas in Lymnaea, only a limited number (50–60) of neurons showed immunoreactivity. The immunostained neurons in Helix were characterized by rich arborizations in all central ganglia and revealed massive innervation of all peripheral nerves and the neural (connective tissue) sheath around the ganglia and peripheral nerve trunks. A small number of Helix nerve cell bodies in the viscero-parietal ganglion complex were also found to be innervated by PDH-like immunoreactive processes. Hence, a complex central and peripheral regulatory role, including neurohormonal actions, is suggested for a PDH-like substance in Helix, whereas the sites of action may be more limited in Lymnaea.
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  • 82
    ISSN: 1432-0878
    Keywords: Key words Hypophysis ; Aromatase ; Immunocytochemistry ; Morphometry ; Gender differences ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract In order to analyze whether aromatase is present in the hypophysis of adult rats, we have performed an immunohistochemical study in young adult male and female rats. Our study has revealed that the hypophysis of adult rats contains aromatase, although marked differences are found between the sexes. The hypophyses of male rats have cells immunoreactive for the enzyme, 34.40% of these hypophyseal cells showing reaction. By contrast, cells from female rats show very little reaction, only 0.84% of them being reactive. No significant differences in the percentage of immunoreactive cells between one phase and another are observed during the estrous cycle. Our results point to the immunohistochemical expression of aromatase in the hypophysis of adult rats and at the same time suggest that its expression is sex-dependent. The enzyme may therefore be involved in the regulation of adenohypophyseal cytology by androgens.
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  • 83
    ISSN: 1432-0878
    Keywords: Key words Melanin-concentrating hormone neurons ; Lateral hypothalamic slice culture ; Immunocytochemistry ; Ultrastructure ; In situ hybridization ; Competitive RT-PCR ; Leptin assay ; Rat (Sprague Dawley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Hypothalamic slices containing the lateral hypothalamic area (LHA) were prepared from 6- to 8-day-old rats and maintained in stationary culture for up to 35 days in order to analyse how well the melanin-concentrating hormone (MCH) neurons survived. As previously reported for other brain areas, this method yielded a long-term well-preserved organotypic organization. Light- and electron-microscopic investigations showed that differentiation continued and that synaptic contacts developed in vitro. After a period of elimination of damaged cells and fibres, most of the remaining neurons and glial cells retained a normal morphology throughout the culture period. MCH neurons, in particular, survived well as attested by the strong immunocytochemical and in situ hybridization signals still observed after several weeks. In a comparison with the day of explantation, competitive reverse transcription/polymerase chain reaction demonstrated the remarkable stability of the level of MCH mRNA at least until the 20th day in culture; after 30 days, the clear decrease in this level seemed to be correlated with a loss of MCH neurons, rather than with a decrease in MCH expression. After 10 days of culture, the incubation of slices in the presence of the hormone leptin (50 ng/ml) resulted in a strong decrease of MCH gene expression, suggesting that MCH neurons retained their physiological properties. Thus, the LHA slice stationary culture, especially between one and three weeks (i.e. after tissue stabilization and before extensive cell loss), appears to be a suitable method for physiological and pharmacological studies of these neurons.
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  • 84
    ISSN: 1432-0878
    Keywords: Key words Caveolin ; Caveolae ; Lung ; Alveolar epithelial type I cell ; Immunocytochemistry ; Electron microscopy ; Confocal laser scanning microscopy ; Rat (CD)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.
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  • 85
    ISSN: 1615-6102
    Keywords: Cumulus oophorus ; Ovarian follicle ; Fertilization ; Ultrastructure ; Immunocytochemistry ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The fine structure of the human cumulus oophorus has been reviewed on the basis of scanning and transmission electron microscopic observations as well as of immunofluorescence data. Tissues sampled from preovulatory ovarian follicles and cumulus-enclosed oocytes and fertilized eggs (collected from the oviduct or obtained during in vitro fertilization procedures) have been evaluated from a microtopographic and morphodynamic point of view in order to better clarify the possible role of this population of cells. In particular, the following aspects have been studied and discussed: the presence of multiple close contacts (modulated by the interposition of the zona pellucida) between the oocyte surface and the long microvillous evaginations projecting from the inner aspect of corona cells surface (through these structures the intraovarian cumulus oophorus may control oocyte growth and metabolism up until the time of ovulation); the occurrence of different subpopulations of cells (steroid-synthetic cells, cells producing adhesive proteins, leukocytes, macrophages) in the postovulatory, extraovarian cumulus oophorus surrounding oocytes, zygotes and early developing embryos. All these elements found in the cumulus mass may positively act, through their paracrine activities, on the chemical composition of the microenvironment in which fertilization occurs.
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  • 86
    ISSN: 1615-6102
    Keywords: Haustorium ; Immunocytochemistry ; Interface ; Parasitism ; Defense mechanisms ; Scrophulariaceae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The haustorial structure of three African parasitic members of the family Scrophulariaceae (Buchnera hispida, Rhamphicarpa fistulosa, andStriga hermonthica) has been studied with regard to the interface between haustoria and the invaded host roots. Immunocytochemical observations at the light and electron microscopical level were carried out with monoclonal antibodies against pectin. JIM5, JIM7, and hydroxyproline-rich glycoprotein (HRGP), LM1. Lignins have been visualized by phloroglucinolhydrochloric acid staining. At the margin of the lateral interface (contact area of host root cortex and parasite cells), JIM5- and JIM7-labelled substances accumulate between parasite papillae and the host root surface indicating that pectins are implicated in sealing the parasite to the attacked host organ. The lateral interface is characterized by the presence of compressed, necrotic host cells, whereas the central interface (contact area between host stele and parasite cells) is generally devoid of host cell remnants. Phenolic substances and/or lignins can be found at the site of penetration of the haustorium into the host root. These observations and the fact that HRGPs accumulate at the host side of the interface support the view of, at least, a partial defense reaction in the invaded host root tissues. Within haustoria, HRGPs were restricted to differentiating xylem elements, implying a spatio-temporal regulation of HRGPs in developmental processes.
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  • 87
    ISSN: 1615-6102
    Keywords: Flow cytometry ; Propidium iodide ; Cell cycle analysis ; Bromodeoxyuridine ; DNA synthesis ; Immunocytochemistry ; Glomus versiforme
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The cell cycle of an arbuscular mycorrhizal fungus,Glomus versiforme, was determined by flow cytometric analysis of nuclei isolated from spores and mycorrhizal roots of leek, and by immunogold staining after bromodeoxyuridine (BrdU) uptake by DNA. The aims of our work were to establish: (i) whether there are changes in ploidy during fungal growth and morphogenesis, (ii) when and where the cell cycle is activated. Our results demonstrate that nuclei isolated from quiescent spores ofG. versiforme are arrested in the GO/G1 phase (99.2%), whereas fungal nuclei from mycorrhizal roots are in the synthetic (S) (10.1%) and G2/M phase (3.9%). Nuclei undergoing DNA synthesis were detected in situ after BrdU uptake. Labelled nuclei were observed in intercellular hyphae and in large arbuscular trunks. This paper demonstrates that colonization of an arbuscular mycorrhizal fungus is linked to activation of its cell cycle.
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  • 88
    Electronic Resource
    Electronic Resource
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    Protoplasma 186 (1995), S. 169-182 
    ISSN: 1615-6102
    Keywords: Acetylated tubulin ; Tyrosinated tubulin ; Taxol ; Pteridophyte ; Spermatogenous cells ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Acetylation and tyrosinization are post-translational modifications of tubulin generally associated, respectively, with highly stable or dynamic microtubule arrays in animals and protists. Little is known of these modifications in land plants, however. We examined the presence and distribution of post-translational tubulin modifications in developing spermatogenous cells of the pteridophyteCeratopteris richardii by immunofluorescence and immunogold, utilizing antibodies specific for acetylated and tyrosinated tubulin. Acetylated tubulin is found in mid to late stage spermatogenous cells in stable microtubule configurations: the spline, flagella, and basal bodies. Tyrosinated tubulin, a modification associated with dynamic microtubule arrays, is also present in these structures as well as all other microtubules in the cell. The lamellar strip of the multilayered structure, a body previously described as tubulin-containing, was not labelled by any of the tubulin antibodies or antiserum. Treatment of cultures with the microtubule stabilizer taxol results in the appearance of new arrays of microtubules, including bundles in the cytoplasm. Only those new taxol-induced microtubule arrays present in mid to late stage cells (i.e., those with other normally acetylated tubulin arrays) have acetylated domains. Younger spermatogenous cells had similar microtubule bundles but no acetylated tubulin. Tyrosinated tubulin was found in all these taxol-stabilized arrays. These data indicate that, although these pteridophyte cells have the ability to acetylate tubulin, that this ability is limited to stages after the final spermatogenous cell mitosis and is limited to the highly stable spline and flagella microtubules.
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  • 89
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 206 (1999), S. 1-10 
    ISSN: 1615-6102
    Keywords: Arabinogalactan protein ; Cell wall component ; Immunocytochemistry ; Pectin ; Pinus densiflora ; Pollen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In order to compare cell wall formation in gymnosperm pollen with that in angiosperm pollen, the distribution of cell wall constituents in the pollen grain and pollen tube ofPinus densiflora was studied immunocytochemically with monoclonal antibodies JIM 5 (against non- or poorly esterified pectin), JIM 7 (against highly esterified pectin), JIM 13 (against arabinogalactan proteins, AGPs), and LM 2 (against AGPs containing glucuronic acid). In the pollen grain wall, only the outer layer of the intine was labeled with JIM 5 and weakly with JIM 7. The tube wall was scarcely labeled with JIM 5 and very weakly labeled with JIM 7. In contrast, the whole of both the intine and the tube wall was strongly labeled with JIM 13 and LM 2, and the generative-cell wall was also labeled only with LM 2. The hemicellulose B fraction, which is the main polysaccharide fraction from the pollen tube wall, reacted strongly with JIM 13 and especially LM 2, but not with antipectin antibodies. These results demonstrate that the wall constituents and their localization inP. densiflora pollen are considerably different from those reported in angiosperm pollen and suggest that the main components of the cell wall ofP. densiflora pollen are arabinogalactan and AGPs containing glucuronic acid.
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  • 90
    ISSN: 1615-6102
    Keywords: Floral binding protein ; Flower development ; Immunocytochemistry ; MADS-box genes ; Ovule ; Transcription factors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary DuringPetunia hybrida seed development, the MADS-box genes encoding the floral binding proteins (FBP) 7 and 11 are expressed in the seed coat and not in the endosperm or embryo. These proteins are thought to function as transcription factors and are essential for ovule formation inPetunia spp. Immunocytochemical methods were used to analyze the distribution of FBP7 and FBP11 after fertilization in wild type and ectopic and cosuppression mutants. During the first nine days of seed development the protein was found in the nuclei of seed coat cells, of both wild-type plants and plants which ectopically expressedFBP11. The signal for FBP7 and -11 proteins diminished during seed development, was first lost in the outer epidermis of the seed coat, then in the endothelium, and finally, at 9 days after pollination (DAP), the protein could not be detected anymore in the parenchyma cells of the seed coat. Although the distribution patterns in wild-type andFBP11 ectopically expressing plants are similar, the latter exhibited higher protein levels. A mild-cosuppression mutant ofFBP7 andFBP11, having only a total of 5%FBP7 and -11 mRNA, showed hardly any FBP7 and -11 proteins. The lack of FBP7 and -11 caused endosperm degeneration in the mutant at a moment when the protein had already decreased to an undetectable level in the wild type and ectopic expression mutant (i.e., at 13 DAP). It is suggested that till about 9 DAP a minimal amount of FBP7 and -11 is needed for the normal functioning of the seed coat during later stages, i.e., for transfer of nutrients to endosperm and embryo. Besides the immunocytochemical data on theFBP7 andFBP11 MADS-box gene products, the morphological analysis of wild type and mutants contributes details on early seed development inPetunia hybrida.
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  • 91
    ISSN: 1435-604X
    Keywords: Heat shock protein 70 (hsp70) ; Carbon dioxide laser ; Sodium arsenite ; Human diploid fibroblast ; Electrophoresis ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Physics , Technology
    Notes: Abstract In a previous study, it was demonstrated that heat shock protein 70 (hsp70) was induced by short duration (〈1 s) carbon dioxide (CO2) laser radiation (10.6Μm. To further characterize the stress response after laser irradiation, the time course of synthesis and cellular localization of hsp70 has been followed. As it had been shown that laser irradiation elevated the temperature to about 67±2
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