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  • 11
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Monoclonal and polyclonal antibodies to neurofilament proteins, neuron-specific enolase, glial fibrillary acidic protein and S-100 have been used to demonstrate nerves, ganglion cells and the supportive glial system of the innervation of various organs. The female genitalia, the urinary tract, the respiratory system, the pancreas, the heart and the skin of several mammalian species, including rat, mouse, guinea pig, cat, pig, monkey and man were fixed in parabenzoquinone and portions of each organ were snap frozen. Serial or free-floating thick cryostat sections were stained using indirect immunofluorescence and peroxidase anti-peroxidase immunocytochemistry. In addition, the newly described and highly sensitive immunogold-silver staining technique was used on Bouin's-fixed and wax-embedded tissues. Antibodies to neurofilament proteins seemed to react with neuronal structures in all the species studied. Alternately stained serial sections showed a similar distribution of neurofilament proteins and neuron-specific enolase-containing nerves. Neuron-specific enolase staining had a diffuse appearance and was found to be highly variable, indicating that the neuron-specific enolase content might be related to the physiological state of the nerves and ganglion cells, whereas antibodies to neurofilament protein gave a consistently intense and very clear picture of the ganglion cells and nerve fibres. Antibodies to S-100 stained supportive elements of the peripheral nervous system in all tissues examined, whereas antibodies to glial fibrillary acidic protein were more selective.
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  • 12
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The role of regulatory peptides and the existence of specific peptide receptors are becoming established. However, techniques for the ultrastructural localisation of these receptors are fraught with difficulties. We propose here a novel technique for receptor localisation using a dimerie peptide ligand and electron microscopical immunocytochemistry. The dimeric ligand is used as a bridge between the receptor and a specific anti-ligand antibody. By this method we have localised receptors for bombesin in cells of small cell lung carcinoma in culture. The results support previous biochemical evidence for the existence of said receptors and the technique should be applicable for the localisation of other receptors recognising small ligands.
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  • 13
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The morphological relationship between sensory and sympathetic nerves was studied in tissues of the eye and the oral cavity following chronic sympathetic or sensory denervation. Immunoreactivities for calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH) were used as indexes to assess the changes of the two nerve populations after denervation. Following surgical sympathectomy, a marked increase of CGRP-containing fibres was seen in all tissues studied, while TH-imunoreactive fibres were totally depleated. Conversely, after capsaicin treatment, an increase of TH-immunoreactive nerves was found in the same tissues, concomitant with a sharp decrease of CGRP-immunoreactive nerves. These changes were particularly evident in iridial stroma and around blood vessels in all tissue, where sensory and sympathetic nerves have a closely overlapping distribution pattern. The altered proportion of sensory peptide-and catecholamine-containing nerves following sympathetic and sensory denervation suggest that there is a reciprocal trophic influence between the two nerve subsets, possibly with the intervention of neurotrophic substances such as nerve growth factor. These results indicate a close interaction between sensory peptidergic and sympathetic nervous systems in peripheral organs.
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  • 14
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The distribution and concentrations of neuropeptide Y (NPY) in kidneys, renal arteries, heart, aorta, mesenteric artery and adrenal glands from aorta-ligated hypertensive rats were studied by immunocytochemistry and radioimmunoassay. Immunocytochemistry showed that in the hypertensive animals NPY-immunoreactive fibres were decreased in both kidney and renal artery, above and below the ligation, and in mesenteric arteries. The depletion of NPY-containing nerves in the kidney was more pronounced around the juxtaglomerular apparatus than in other areas of the organ. By radioimmunoassay, the concentrations of NPY immunoreactivity were significantly lower in the hypertensive animals when compared with the controls, (kidney: hypertensive 1.0±0.1; controls 2.0±0.2 pmol/g, mean±SEM; p〈0.05 renal artery: hypertensive 5.0±0.8; controls 12.1±2.0; p〈0.05 and mesenteric artery: hypertensive 8.6±1.9; 17.6±3.0; p〈0.01). While there were no statistically significant changes in the levels of NPY immunoreactivity in the other areas studied, there was a general trend for the level to fall in the renal artery below the ligation (hypertensive 10.6±1.5; control 15.3±2.4; p〉0.05). It is of interest that changes were observed in the vasoconstrictor peptide NPY in this commonly used model of hypertension.
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  • 15
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The guinea pig uterus is supplied by different populations of nerves which can be demonstrated by specific immunocytochemical and histochemical techniques. So far, there has been no single marker displaying entire peripheral innervation patterns. Recently, protein gene product (PGP) 9.5, a cytoplasmic protein in neurons and neuroendocrine cells, was found to visualize both different populations and subtypes of nerves. This prompted the present study of using PGP 9.5 for visualization of the whole uterine innervation. This was performed by the indirect immunofluorescence method using antiserum to PGP 9.5 raised in rabbits. PGP-immunoreactivity was present in all neuronal parts of the extrinsic and intrinsic uterine innervation, including different subpopulations of nerves. This was verified by chemical sympathectomy and sensory denervation with 6-hydroxydopamine and capsaicin-treatment respectively, and double immunostaining. By term a disappearance of uterine PGP-nerve-immunoreactivity was observed which was almost complete in fetus-bearing uterine tissue and further strengthens previous assumptions of a general, pregnancy-induced uterine neuronal degeneration. The developmental time-course and morphology of PGP-immunoreactive nerve structures was similar to that for other neuronal markers and support the suggestion of PGP-immunoreactivity as a general marker for the entire uterine innervation, and suggests that the presence of PGP 9.5-immunoreactivity may coincide with functional maturation of uterine innervation.
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  • 16
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The ontogenetic development of the guinea pig uterine autonomic innervation was studied immunohistochemically using neurofibrillary protein (NF) and neuron specific enolase (NSE) as general neuronal markers, tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) as specific markers for adrenergic innervation and S-100 protein as marker for Schwann cell structure and/or function. In addition, comparisons were made of the development of the different populations of peptide-containing nerves. The structure and time of appearance were similar for nerves with NF-, NSE-, TH- and DBH-immunoreactivities, which were first present in the organ periphery as coarse nerve trunks, then extending centrally and branching into non-varicose nerves. From these, varicose nerves developed first in relation to vessels and then in association with the myometrial smooth musculature. Development was completed carlier in the cervix than in the uterine horns suggesting differences in local environment. In comparison, S-100 nerve-immunoreactivity appeared later but attained complete development more rapidly than axonal structures. Neuropeptide Y-immunoreactive nerves showed a similar developmental pattern to presumed adrenergic nerves, further verifying the assumption of intraneuronal localization of NPY in uterine adrenergic nerves. Other peptide-containing nerves were developed later probably reflecting differences in neuronal growth properties.
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  • 17
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    Electronic Resource
    Springer
    Histochemistry and cell biology 89 (1988), S. 481-483 
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In situ hybridisation detection of mRNAs using riboprobes has become a widely used technique. However, the identification of cells producing closely-related yet distinct mRNAs is difficult with the usual size probes. More-over, it is not always easy to obtain the required cDNA essential for cRNA probe synthesis. To avoid these problems, we have used synthetic oligodeoxynucleotides to generate short, single stranded RNA probes (“oligo-riboprobes”). These probes can be labelled to very high (109 cpm/μg) specific activity and can be prepared for any published nucleotide sequence. We have used these probes to localise β (preprotachykinin) PPT mRNA producing neurons in rat hypothalamus and bowel. The results were compared to that obtained with cRNA probes generated from β preprotachykinin cDNA.
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  • 18
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The presence of bombesin (gastrin-releasing peptide, GRP)-like immunoreactivity in mucosal endocrine cells of human fetal lung is well established. In this study we have investigated the localisation of pro-GRP mRNA and GRP gene products and compared the distribution and levels of extractable GRP-and C-terminal flanking peptide of human pro-GRP-like immunoreactivity in order to verify synthesis and to investigate their coexistence and molecular forms. Human fetal lungs (14 to 23 weeks gestation) were immunostained, and extracts were assayed using regionspecific antisera to pro-GRP. Additional antisera to chromogranin and protein gene product 9.5 (PGP 9.5) were used for immunostaining by the peroxidase anti-peroxidase technique and for double immunofluorescence staining using antisera raised in two species. Immunoreactivity for both bombesin (GRP) and flanking peptide was seen mainly in the same endocrine cells, but more cells were stained with antisera to flanking peptide than with antiserum to bombesin (GRP). In situ hybridisation showed that pro-GRP mRNA was present and thus synthesis of the peptides was taking place. Endocrine cells and nerve fibres were PGP 9.5-immunoreactive, and a subset of cells was immunoreactive for bombesin gene products. Radioimmunoassay and chromatography show that pro-GRP is present in both the uncleaved and cleaved forms, and, in agreement with immunocytochemistry results, that an excess of C-terminal peptide of pro-GRP is detectable. It is therefore concluded that GRP-like peptides and flanking peptide are co-local-ised in human pulmonary endocrine cells, but the latter is found in larger concentrations than free GRP. Thus GRP-like peptides may be secreted separately from the flanking peptide(s) of pro-GRP. Furthermore PGP 9.5 appears to be a useful marker for endocrine cells in the respiratory epithelium of human fetal lung.
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  • 19
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary GAWK is a recently discovered peptide isolated from extracts of human pituitary gland and subsequently shown to be identical to sequence 420–493 of human chromogranin B. The distribution of this peptide was studied in human gut, pancreas, adrenal and pituitary glands using antisera to two portions of the 74 amino acid peptide (sequences 1–17 and 20–38). In addition, the co-existence of GAWK immunoreactivity with other peptides and chromogranin B was investigated using comparative immunocytochemistry. In the gut, GAWK was localised mainly to serotonin-containing cells of the mucosal epithelium, where electron microscopy showed it to be stored in typical electron-dense (250 nm diameter) granules, and to a moderate population of nerve fibres in the gut wall. Considerable quantities of GAWK-like immunoreactivity were measured in the gut, up to 36.3±18 pmol GAWK 1–17/g wet weight of tissue (mean±SEM) and 12.4±2.9 pmol GAWK 20–38/g. Chromatography of gut extracts revealed several GAWK-like immunoreactive peaks. GAWK-like immunoreactivity was also detected in endocrine cells of pancreas, pituitary gland and adrenal medulla, where the highest concentrations of GAWK-like immunoreactivity were measured (GAWK 1–17 2071.8±873.2 and GAWK 20–38 1292.7±542.7 pmol/g). Endocrine cells containing GAWK-like immunoreactivity were found also to be immunoreactive for chromogranin B. Our results define a discrete distribution of GAWK immunoreactivity in human endocrine cells and nerves and provide morphological support for the postulated precursor-product relationship between chromogranin B and GAWK. Details of the functions of this peptide are awaited.
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  • 20
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The identity of galanin- and vasoactive intestinal polypeptide-(VIP) immunoreactive (IR) cells in the rat anterior pituitary was investigated using immunocytochemistry and, since levels of both peptides are stimulated by oestrogen, the effect of oestrogen treatment and gonadectomy on the expression of both peptides was examined. In normal male rats, few galanin-IR and very few VIP-IR cells were found. Colocalisation studies performed on 2-μm serial paraffin sections revealed that in these animals galanin IR was present in somatotrophs and thyrotrophs. In normal females in dioestrus many lactotrophs, in addition to somatotrophs and thyrotrophs, expressed galanin, but very few VIP-IR cells were seen. In cryostat sections of normal rat pituitaries, slightly more VIP-IR cells were present. Oestrogen treatment in females produced an increase in frequency of galanin-IR cells, the vast majority of which were lactotrophs, and more VIP-IR cells, identified as lactotrophs, also appeared. VIP was present in a subset of galanin-IR lactotrophs after oestrogen treatment. After ovariectomy female pituitaries resembled those of normal males, with few galanin positive cells none of which were lactotrophs, and hardly any VIP-IR cells. Thus these two peptides are present in specific endocrine cell types of rat anterior pituitary and display plasticity of expression in different cell types under the influence of oestrogen. Their roles in control of pituitary hormone secretion are supported by these findings, and it is possible that both peptides act in a paracrine fashion within the pituitary.
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