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  • 1
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary We have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a 32P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.
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  • 2
    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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  • 3
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Several studies have shown the use of non-radioactive labelled DNA probes for in situ hybridisation, mainly to identify cellular DNA. In this study mRNA in situ hybridisation was performed on rat pituitary with biotinylated complementary (c) RNA probes for rat prolactin and growth hormone (GH), and compared with radioactive 35S-radiolabelled probes. Biotinylated cRNA probes were labelled with either biotin-11-UTP or with allylamine-UTP, the latter method being able to produce a higher yield of labelled RNA. Different detection systems were tested, and hybridisation signal was seen in cells of anterior pituitary with both types of biotinylated probes. The signals were detected using either avidin-biotin-complex with peroxidase (ABC), peroxidase-anti-peroxidase (PAP) or gold-silver methods. ABC peroxidase detected using glucose oxidase-diaminobenzidine (DAB)-nickel solution appeared to be the best method for detecting labelled RNA probes, with very strong signal and low background. The biotinylated probes were comparable in sensitivity to the radiolabelled probes in detecting prolactin and GH mRNAs in the anterior lobe of the rat pituitary. These results indicate an alternative methods of labelling and detection of biotinylated probes which could have a potential role in research and diagnostic techniques.
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  • 4
    Electronic Resource
    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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  • 5
    ISSN: 1432-2307
    Keywords: Bombesin ; Human pro-bombesin ; Lung ; Small cell carcinoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Small cell carcinoma of the lung is a highly malignant tumour. Its known biological products which include bombesin, do not allow the prediction of tumour behaviour. Molecular biology has revealed the amino acid sequence of human pro-bombesin, which consists of a signal peptide, the bioactive bombesin molecule and a C-terminal peptide. We have raised a rabbit antiserum to the first (N-terminal) 21 amino acids of the predicted C-terminal peptide. A total of 505 (361 neuroendocrine) surgically resected pulmonary tumours were evaluated for the presence of immunoreactive bombesin and C-terminal peptide. Strong immunostaining was obtained with the antiserum to the C-terminal peptide of human probombesin in 70% of the small cell carcinomas (175/250), in 63% of atypical (aggressive) carcinoids (31/49) but only in 16% of benign carcinoids (10/62). In contrast, bombesin immunostaining was focal and only moderately strong and the relative proportion of positive cases was quite evenly distributed amongst the neuroendocrine tumours: 35% of carcinoids (22/62), 22% of atypical carcinoids (11/49) and 25% of small cell carcinoma (62/250). None of the squamous, adeno, or large cell undifferentiated carcinomas were immunoreactive for bombesin or the C-terminal peptide. Radioimmunoassay and chromatography of extracts of tumours recovered from wax blocks revealed high concentrations of C-terminal peptide immunoreactivity (241±66 pmol/g of tissue) in all 12 small cell carcinomas studied, moderate concentrations in carcinoid tumours (50±7 pmol/g) and none in non-small cell carcinomas. Patients with tumours showing immunoreactivity to the C-terminal peptide of human pro-bombesin had a significantly shorter survival time than those without immunoreactive peptide (185±16.49 days, mean± SEM, and with 1128±226 days, respectivelyP〉 0.02). The apparent presence of the C-terminal peptide of human pro-bombesin in higher concentrations than bombesin in the more malignant class of endocrine tumours, mainly small cell carcinomas associated with the poorest prognosis, suggests that the antiserum to this C-terminal peptide is not only a useful pathological marker but may prove to be of value in investigating the biological behaviour of small cell carcinomas and predicting the clinical course of the disease.
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  • 6
    ISSN: 1432-2307
    Keywords: Cystitis ; Metaplasia ; Endocrine cells ; Urinary bladder
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Several types of metaplasia can occur in human bladder epithelium under certain pathological conditions. We investigated 65 cases of cystitis, associated with different types of metaplasia, for the presence of endocrine cells, using histochemical and immunocytochemical methods. Tissues were obtained at cystoscopy and were routinely fixed in 10% buffered formalin. Endocrine cells were demonstrated, between the epithelial cells, in 40 out of 50 cases of cystitis glandularis or cystica. These cells were positive by the Grimelius' silver impregnation technique and were immunoreactive for protein gene product (PGP 9.5), a new general neuroendocrine marker, chromogranin and serotonin. No endocrine cells were detected in any of the specimens of normal epithelium nor those showing squamous metaplasia. Eighteen of these cases showed prominent nerve bundles in the sub-epithelial tissue, as revealed by PGP immunoreactivity.
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  • 7
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We have examined the effect of prolonged treatment with topical corticosteroid on allergen-induced early and late nasal responses and the associated inflammatory cell infiltrate in grass pollen sensitive allergic rhinitics. Following a randomized double-blind 6 week treatment period with fluticasone propionate 200 μg aqueous nasal spray twice daily or matched placebo spray, nasal provocation was performed using Timothy grass pollen extract. Nasal symptoms were recorded at intervals from 0 to 24 h. Nasal biopsies were performed before treatment and at 24 h after allergen and processed for immunohistology. When corticosteroid-treated patients were compared with the placebo group there was an approximately 50% decrease in the size of the early (0-60 min) response and almost complete inhibition of late (1–24 h) nasal symptoms after allergen challenge. After allergen challenge markedly fewer T lymphocytes and CD25+ (interleukin-2 receptor bearing) cells were observed in both the epithelium and submucosa in fluticasone treated patients compared with the placebo group. Significantly less total and activated eosinophils were observed, particularly within the nasal epithelium. Submucosal mast cell counts were decreased, whereas increased numbers of submucosal neutrophils were observed. These results confirm that topical corticosteroid treatment inhibits allergen-induced early and late nasal responses. This may possibly occur following a decrease in T lymphocytes and/or mast cells and their products and a consequent reduction in tissue eosinophilia.
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  • 8
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background Asthmatic airways are characterized by infiltration with a variety of inflammatory cells such as mast cells and eosinophils. Stem cell factor (SCF) is an important activating and chemotactic factor for both mast cells and eosinophils. In addition, it is a critical growth and differentiation factor for mast cells.Objectives To investigate the contribution of SCF to the pathogenesis of asthma, we examined the expression of SCF and its receptor c-kit in bronchial biopsies and bronchoalveolar lavage (BAL) specimens obtained from asthmatic subjects (n=13) and non-asthmatic control subjects (n=10).Methods SCF and c-kit were detected by in situ hybridization (ISH) and immunocytochemistry (ICC). In order to phenotype the cells expressing SCF and c-kit in asthmatic tissue and BAL cells, combined ISH and ICC were also performed.Results There was a significant difference (P〈0.001) in the SCF mRNA expression in asthmatic airway epithelium (70.38±12.33% positive cells) compared with controls (12.7±17.21% positive cells). There was also a significant difference in subepithelial SCF-mRNA expression, being higher in asthmatics (P〈0.001). A significant difference was also found in c-kit receptor mRNA expression in asthmatic biopsies both in epithelium (P〈0.001) and subepithelium (P〈0.05) compared with controls. ICC results were consistent with the ISH for both SCF and c-kit receptor from asthmatics and controls. The SCF and c-kit receptor mRNA and immunoreactivity in cells recovered from bronchial washing were also significantly higher in asthmatics compared with controls (P〈0.05). While SCF expression was localized predominantly in the epithelial layer in bronchial biopsy tissues, alveolar macrophages were found to be the major source of SCF in bronchial washing from asthmatic subjects.Conclusion The results of this study demonstrate the increased expression of SCF and its receptor, c-kit within human asthmatic airways, which suggests an important role of this cytokine in the pathophysiology of asthma.
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  • 9
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background In bronchial mucosa, T cells are in close association with fibroblasts. This cell contact raises the possibility of cross-talk between the two cell types through cytokines, such as interleukin-4 (IL-4).Objective We postulated that IL-4 may modulate collagen synthesis and degradation in the fibroblasts of asthmatics.Methods Bronchial fibroblasts from asthmatics (BAF) and normal controls (BNF) were stimulated with IL-4. Procollagen I gene expression and protein production were measured by real-time PCR, RT-PCR, and radioimmunoassay. The effect of IL-4 on the regulation of procollagen I (α1) promoter was studied through transient cell transfections. The implication of Sp1 and AP-1 in regulating IL-4-induced procollagen I (α1) production was determined. The effect of IL-4 on metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) production and gene expression was evaluated.Results Following IL-4 stimulation, there was a significant increase in the expression of mRNA of procollagen I (α1) by human bronchial fibroblasts of asthmatics and controls. IL-4 has a dose–response effect on mRNA, with a maximal effect at 5 ng/mL, as determined by real-time PCR. The maximal increase in procollagen I (α1) was observed at 6 h after IL-4 stimulation in both BNF and BAF. BAFs have a greater increase in the procollagen I (α1)/β2 microglobulin ratio after 6 h of IL-4 stimulation (4.1×10−2±0.03 to 20.8×10−2±0.1) compared with BNF (2.9×10−2±0.006 to 9.2×10−2±0.08) (P=0.001). In transient transfection experiments, IL-4 increased promoter activity by threefold in BAF and BNF. Sp1 was up-regulated after IL-4 stimulation and AP-1 was down-regulated as shown by electrophoretic mobility shift assay. IL-4 decreased MMP-2 protein and mRNA levels, and did not alter TIMP-2 production.Conclusions IL-4 positively regulates procollagen I (α1) transcription by direct promoter activation and increases the TIMP-2/MMP-2 ratio, thereby supporting the profibrotic effect of this cytokine. Thus, this study emphasizes that IL-4 may be considered as a link between inflammation and collagen deposition observed in asthmatic airways.
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Histopathology 23 (1993), S. 0 
    ISSN: 1365-2559
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Twenty-two neuroendocrine tumours of the larynx were investigated using a panel of immunocytochemical markers. Three were small cell carcinomas, eight were large cell neuroendocrine carcinomas and 11 were paragangliomas. Twenty were positive for protein gene product 9.5, 19 for neuron-specific enolase, 15 for chromogranin A, nine for bombesin, eight for substance P, eight for neuropeptide Y, eight for metenkephalin, seven for somatostatin, five for calcitonin, eight for calcitonin gene-related peptide and one for vasoactive intestinal polypeptide. Bombesin immunoreactivity was largely restricted to the small cell carcinomas and large cell neuroendocrine carcinomas and neuropeptide Y, metenkephalin and substance P to the parangangliomas. This comprehensive immunocytochemical analysis of neuroendocrine tumours of the larynx demonstrates that these tumours represent special entities but have similar patterns of immunostaining to those of neuroendocrine tumours in other sites.
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