The Use of Antiavidin Antibody and Avidin-Biotin-Peroxidase Complex in Immunoperoxidase Technics

Hsu, Su-Ming; Raine, Laurence; Fanger, Herbert

doi:10.1093/ajcp/75.6.816

Cited by 965 publications

(237 citation statements)

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“…NOS protein was detected by anti-endothelial NOS mAb (1:250 dilution) and biotinconjugated anti-mouse IgG (1:500) as the secondary antibody followed by staining with horseradish peroxidase-linked streptavidin and visualized by light microscopy as described (25).…”

Section: Methodsmentioning

confidence: 99%

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

Belting

Mani

Jönsson

et al. 2003

Journal of Biological Chemistry

151

113

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Polyamines (putrescine, spermidine, and spermine) are essential for growth and survival of all cells. When polyamine biosynthesis is inhibited, there is up-regulation of import. The mammalian polyamine transport system is unknown. We have previously shown that the heparan sulfate (HS) side chains of recycling glypican-1 (Gpc-1) can sequester spermine, that intracellular polyamine depletion increases the number of NO-sensitive N-unsubstituted glucosamines in HS, and that NO-dependent cleavage of HS at these sites is required for spermine uptake. The NO is derived from S-nitroso groups in the Gpc-1 protein. Using RNA interference technology as well as biochemical and microscopic techniques applied to both normal and uptake-deficient cells, we demonstrate that inhibition of Gpc-1 expression abrogates spermine uptake and intracellular delivery. In unperturbed cells, spermine and recycling Gpc-1 carrying HS chains rich in Nunsubstituted glucosamines were co-localized. By exposing cells to ascorbate, we induced release of NO from the S-nitroso groups, resulting in HS degradation and unloading of the sequestered polyamines as well as nuclear targeting of the deglycanated Gpc-1 protein. Polyamine uptake-deficient cells appear to have a defect in the NO release mechanism. We have managed to restore spermine uptake partially in these cells by providing spermine NONOate and ascorbate. The former bound to the HS chains of recycling Gpc-1 and S-nitrosylated the core protein. Ascorbate released NO, which degraded HS and liberated the bound spermine. Recycling HS proteoglycans of the glypican-type may be plasma membrane carriers for cargo taken up by caveolar endocytosis.

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Section: Methodsmentioning

confidence: 99%

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

Belting

Mani

Jönsson

et al. 2003

Journal of Biological Chemistry

151

113

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“…Immunohistochemical Analysis-The kidneys removed from Sprague-Dawley rats (20 weeks, male) and BALB/c mice (10 weeks, female) were fixed in neutralized 10% (v/v) formalin, embedded in paraffin, and sectioned at 3 m. For immunohistochemistry, these sections were stained by the avidin-biotin-peroxidase complex method (23). The primary and secondary biotinylated antibodies for DCXR staining were the anti-mouse DCXR antibody (10 g/ml in PBS) and a goat anti-rabbit immunoglobulin (IgG) antibody (10 g/ml, Vector Laboratories, Burlingame, CA), respectively.…”

Section: Using the Above Antibodiesmentioning

confidence: 99%

Molecular Characterization of Mammalian Dicarbonyl/l-Xylulose Reductase and Its Localization in Kidney

Nakagawa¹,

Ishikura²,

Asami³

et al. 2002

Journal of Biological Chemistry

146

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In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lungspecific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for ␣-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and L-xylulose, and were inhibited competitively by nbutyric acid. Therefore, the proteins are designated as dicarbonyl/L-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and L-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC 1.1.1.5) are identical to Lxylulose reductase (EC 1.1.1.10), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.

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“…The immunoperoxidase procedures (ABC method) using a monoclonal antibody to CD9 (Novocastra, Newcastle, UK; diluted 1 : 20) was carried out in transbronchial biopsy specimens of seven cases of SCLC and one case of lung adenocarcinoma as previously described (Hsu et al, 1981). For antigen retrieval, sections were pretreated with microwaving for 15 min in 1 mm EDTA (pH 8.0) before incubation.…”

Section: Immunohistochemistrymentioning

confidence: 99%

Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer

et al. 2003

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Small cell lung cancer (SCLC) invades locally and metastasizes distantly extremely early when compared with nonsmall cell lung cancer (NSCLC). The underlying molecular mechanisms, however, have not been elucidated. Accumulating evidence suggests that downregulation of several members of tetraspanins is associated with progression of solid tumors, thus indicating poor prognosis. Here we screened 30 lung cancer cell lines for expression of tetraspanins, CD9, CD63, CD81, CD82, CD151, and NAG-2. Flow cytometry revealed that, among these proteins, CD9 is broadly expressed in NSCLC lines, but is absent or highly reduced in most SCLC lines (Po0.0001). Using the Boyden chamber and videomicroscopic cell motility assays, we showed that stable transfection of CD9 into an SCLC line, OS3-R5, reduced cell motility on fibronectin. Furthermore, by transient transfection of green fluorescent protein (GFP)-tagged CD9 into three other SCLC lines, we observed that SCLC cells expressing GFP-CD9 were uniformly less motile than untransfected cells. CD9 or GFP-CD9 was associated with b1 integrins and distributed at the tumor cell periphery and cell-cell contacts, suggesting that CD9 modifies b1 integrin function to reduce motility. These findings suggest that low expression of CD9 may contribute to the highly invasive and metastatic phenotype of SCLC.

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The Use of Antiavidin Antibody and Avidin-Biotin-Peroxidase Complex in Immunoperoxidase Technics

Cited by 965 publications

References 0 publications

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

Molecular Characterization of Mammalian Dicarbonyl/l-Xylulose Reductase and Its Localization in Kidney

Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer

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