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Berlin Brandenburg

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  • Immunohistochemistry
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  • 1
    Language: English
    In: Translational Research, 2010, Vol.156(2), pp.98-105
    Description: Activating V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and V-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene mutations are important predictive markers for antiepidermal growth factor receptor chemotherapy in colorectal cancer (CRC). However, a rapid and accurate assay for KRAS/BRAF mutation detection from routine pathological specimens is lacking in clinical practice. We applied the cycleave polymerase chain reaction (PCR) method to routine KRAS/BRAF genotyping of CRC patients at our institution from 2001 to 2009. The accuracy of cycleave PCR genotyping was shown by the high concordance with reverse transcriptase-PCR-coupled direct sequencing. KRAS gene mutations were analyzed successfully from small biopsy or cytology specimens. Although some surgical specimens could not be evaluated by cycleave PCR, corresponding biopsy specimens could be used instead. This PCR failure observed for some biopsy specimens may have been a result of the use of formalin fixation, as overfixation of surgical specimens by formalin impaired PCR amplification. In conclusion, cycleave PCR is practically applicable to KRAS/BRAF genotyping using small amounts of biopsied tumor cells. Care must be taken in the selection of pathological specimens for KRAS/BRAF testing.
    Keywords: Medicine
    ISSN: 1931-5244
    E-ISSN: 1878-1810
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  • 2
    In: PLoS ONE, 2014, Vol.9(3)
    Description: Oral phosphate loading and calcitriol stimulate Fibroblast growth factor 23 (FGF23) secretion, but the mechanisms underlying the stimulation of FGF23 remain to be studied. We compared the effect of intravenous phosphate loading with that of oral loading on FGF23 levels in normal and 5/6 nephrectomized uremic rats. Uremic rats (Nx) and sham-operated rats were fed a normal phosphate diet for 2 weeks and then divided into 3 groups: 1) with the same phosphate diet (NP), 2) with a high phosphate diet (HP), and 3) NP rats with intravenous phosphate infusion using a microinfusion pump (IV). Blood and urine were obtained 1 day (early phase) and 7 days (late phase) after the interventions. In the early and late phases, serum phosphate levels and fractional excretion of phosphate (FEP) were comparable in the HP and IV groups in both Sham and Nx rats. Serum phosphate levels in the HP and IV groups were equally and significantly higher than those in the NP group only in the late phase in Nx rats. In the early phase, FGF23 levels were comparable in the NP, HP, and IV groups, but were significantly higher in the HP and IV groups compared to the NP group in the late phase in Nx rats. 1α-hydroxylase and sodium dependent phosphate co-transporter 2a expression levels in the kidney in Nx rats were equally and significantly decreased in the HP and IV groups compared with the NP group, while 24-hydroxylase expression was equally and significantly increased. These results show that chronic intravenous phosphate loading increases bioactive FGF23, indicating that an alternative pathway for FGF23 regulation, in addition to the dietary route, may be present. This pathway is clearer under conditions produced by a kidney injury in which phosphate is easily overloaded.
    Keywords: Research Article ; Biology ; Medicine ; Veterinary Science
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: The Journal of biological chemistry, 25 February 2005, Vol.280(8), pp.6721-30
    Description: Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.
    Keywords: Cytochrome P-450 Cyp11b2 -- Genetics ; DNA-Binding Proteins -- Physiology ; Proteins -- Metabolism ; Transcription Factors -- Physiology ; Ubiquitin-Conjugating Enzymes -- Metabolism
    ISSN: 0021-9258
    E-ISSN: 1083351X
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  • 4
    Language: English
    In: Oncology Reports, November 2009, Vol.22(5), pp.1027-1032
    Description: N-acetylglucosaminyltransferase V (GnT-V) is an enzyme that catalyzes β1-6 branching of N-acetylglucosamine (GlcNAc) on asparagine-linked oligosaccharides of cell proteins. The present study aimed to investigate GnT-V expression and its prognostic significance in epithelial ovarian cancer. GnT-V expression was studied by immunohistochemistry in 83 surgically resected ovarian cancers, and the staining intensity was evaluated. High GnT-V expression in cancer cells was found in 17 (20.5%) of the 83 cases, and was positively correlated with early FIGO staging. In the histological type, mucinous adenocarcinoma showed significantly strong immunostaining compared to the non-mucinous type (P〈0.001). In 36 mucinous tumors, the GnT-V immunostaining score was significantly higher in cancer than in benign and borderline tumors (P〈0.001). NOM-1, a human ovarian mucinous adenocarcinoma cell line, expressed strong GnT-V protein and swainsonine treatment suppressed β1-6GlcNAc branching and reduced migration ability significantly (P〈0.001). These results suggested that GnT-V might be involved in the malignant potential of mucinous ovarian cancer.
    Keywords: Medicine;
    ISSN: 1021-335X
    E-ISSN: 17912431
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  • 5
    Language: English
    In: Dementia and Geriatric Cognitive Disorders, May 2005, Vol.19(5-6), pp.282-288
    Description: We sought to determine the changes in the cholinergic pathways, which project from the nucleus basalis of Meynert (nBM) and travel in the subinsular region, in vascular dementia of the Binswanger type (VDBT) and Alzheimer’s disease (AD). The subinsular regions were examined in 6 autopsied brains with VDBT, 5 brains with AD and 4 control brains without any neurologic diseases. The cholinergic pathway was labeled either by histochemistry for acetylcholine esterase (AChE), a degradatory enzyme of ACh, or by immunohistochemistry for choline acetyltransferase, its synthetic enzyme. The numerical density of nBM neurons did not differ significantly between these groups (163 ± 49 in the VDBT, 105 ± 82 in the AD and 198 ± 76 in the control groups), but with a tendency towards a decrease in the AD group. The subinsular cholinergic fibers were impaired, with relative preservation of the nBM neurons in VDBT, whereas both the subinsular cholinergic fibers and the nBM neurons were degraded in AD. These results indicate that the cholinergic pathway is damaged not only in AD, but also in VDBT, and may further provide a pharmacological basis for treatment with AChE inhibitors in VDBT.
    Keywords: Original Research Article ; Cholinergic Neurons ; Vascular Dementia of the Binswanger Type ; Alzheimer'S Disease ; Nucleus Basalis of Meynert ; External Capsule ; Medicine ; Pharmacy, Therapeutics, & Pharmacology
    ISSN: 1420-8008
    E-ISSN: 1421-9824
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  • 6
    Language: English
    In: Dementia and geriatric cognitive disorders, 2005, Vol.19(5-6), pp.282-8
    Description: We sought to determine the changes in the cholinergic pathways, which project from the nucleus basalis of Meynert (nBM) and travel in the subinsular region, in vascular dementia of the Binswanger type (VDBT) and Alzheimer's disease (AD). The subinsular regions were examined in 6 autopsied brains with VDBT, 5 brains with AD and 4 control brains without any neurologic diseases. The cholinergic pathway was labeled either by histochemistry for acetylcholine esterase (AChE), a degradatory enzyme of ACh, or by immunohistochemistry for choline acetyltransferase, its synthetic enzyme. The numerical density of nBM neurons did not differ significantly between these groups (163 +/- 49 in the VDBT, 105 +/- 82 in the AD and 198 +/- 76 in the control groups), but with a tendency towards a decrease in the AD group. The subinsular cholinergic fibers were impaired, with relative preservation of the nBM neurons in VDBT, whereas both the subinsular cholinergic fibers and the nBM neurons were degraded in AD. These results indicate that the cholinergic pathway is damaged not only in AD, but also in VDBT, and may further provide a pharmacological basis for treatment with AChE inhibitors in VDBT.
    Keywords: Basal Nucleus of Meynert -- Pathology ; Cholinergic Fibers -- Pathology ; Dementia, Vascular -- Pathology
    ISSN: 1420-8008
    E-ISSN: 14219824
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 7
    Language: English
    In: Neuroscience Research, 2006, Vol.56(4), pp.391-399
    Description: The functions of fukutin, a gene product responsible for Fukuyama type congenital muscular dystrophy, still remain unclear, although a relation to the glycosylation of α-dystroglycan is presumed. To investigate the functions of fukutin, immunohistochemistry, examination using cultured astrocytes, enzyme-linked immunosorbent assay (ELISA)-based binding assay and immunoprecipitation were performed using control muscle and central nervous system tissues. Immunohistochemistry showed that α-dystroglycan and fukutin were co-expressed, especially in the glial cytoplasm and glia limitans of the central nervous system. An anti-fukutin antibody added to the culture medium did not bring about any changes in the astrocytes cultured on laminin-coated dishes. Together with the immunohistochemical results, the intracellular function of fukutin is considered. ELISA-based binding assay and immunoprecipitation may suggest the direct binding of fukutin and α-dystroglycan, at least in part. Fukutin seems to bind to both the hypoglycosylated and fully glycosylated form of α-dystroglycan, and seems bind to the core area rather than the sugar chain of α-dystroglycan. Fukutin may directly interact with α-dystroglycan during glycosylation, but further examinations are needed to confirm these details.
    Keywords: Fukutin ; Α-Dystroglycan ; Glycosylation ; Intracellular ; Binding ; Muscular Dystrophy ; Medicine ; Anatomy & Physiology
    ISSN: 0168-0102
    E-ISSN: 1872-8111
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  • 8
    Language: English
    In: Developmental Dynamics, November 2004, Vol.231(3), pp.518-526
    Description: The recent discovery of the gene (DM domain gene on Y chromosome and one of the family genes) as a key determinant of male development in the medaka () has led to its designation as the prime candidate gene for sex‐determination in this species. This study focused on the sites and pattern of expression of and genes during gonadal differentiation of medaka to further determine their roles in testis development. mRNA and protein are expressed specifically in the somatic cells surrounding primordial germ cells (PGCs) in the early gonadal primordium, before morphological sex differences are seen. However, somatic cells surrounding PGCs never express during the early migratory period. Expression of persists in Sertoli cell lineage cells, from PGC‐supporting cells to Sertoli cells, indicating that only ‐positive cells enclose PGCs during mitotic arrest after hatching. is expressed in spermatogonium‐supporting cells after testicular differentiation (20–30 days after hatching), and its expression is much higher than that of in mature testes. In XX sex‐reversed testes, is expressed in the Sertoli cell lineage, similar to the expression of in XY testes. These results suggest strongly that regulates PGC proliferation and differentiation sex‐specifically during early gonadal differentiation of XY individuals and that regulates spermatogonial differentiation. Developmental Dynamics 231:518–526, 2004. © 2004 Wiley‐Liss, Inc.
    Keywords: Dmy ; Dmrt1 ; Sex Determination ; Sex Differentiation ; Primordial Germ Cell ; Sertoli Cell ; Medaka
    ISSN: 1058-8388
    E-ISSN: 1097-0177
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  • 9
    In: Pigment Cell Research, April 1996, Vol.9(2), pp.72-76
    Description: Immunohistochemical localization of tyrosinase was examined with a monoclonal antibody (MoAb MAT‐1) against human tyrosinase on routine formalin‐fixed paraffin‐embedded sections of 3 normal skin specimens, 15 melanocytic tumors (6 pigmented nevi, 3 juvenile melanomas and 6 malignant melanomas) and 3 non‐melanocytic tumors. In the melanotic melanomas, almost all tumor cells were clearly stained with the antibody. In the nevocytic nevi, the nevus cells in lower epidermis and upper dermis were positive for MoAb MAT‐1, but negative in middle and lower dermis. All three juvenile melanomas, one amelanotic melanoma, and three non‐melanocytic tumors were entirely negative for MoAb MAT‐1. Thus, MoAb MAT‐1 could recognize the cells with melanogenic activity on routine formalin‐fixed paraffin‐embedded sections. However, the staining quality was not adequate for normal epidermal melanocytes, indicating that small technical innovations in the immunostaining process such as formalin fixation after PBS washing are required. Nevertheless, MoAb MAT‐1 can be expected to be very useful for identifying melanogenic cells on paraffin‐embedded sections, because we have to date no other antibody available for it.
    Keywords: Tyrosinase ; Immunohistochemistry ; Melanoma ; Melanogenesis ; Monoclonal Antibody
    ISSN: 0893-5785
    E-ISSN: 1600-0749
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