[11] Radioiodination of proteins by the use of the chloramine-T method

McConahey, Patricia J.; Dixon, Frank J.

doi:10.1016/s0076-6879(80)70050-2

Cited by 219 publications

(96 citation statements)

References 6 publications

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“…There were no significant changes in surface levels of VLA-5 in these transfectants (data not shown). Contrary to our expectation, cells expressing the Gly 37 , but not Cys 40 mutant showed the levels of adhesion equivalent to, or more than, cells expressing Ha-Ras , whereas those of adhesion of mast cells expressing the Ser 35 , Glu 38 , or Cys 40 mutants were reduced considerably (Fig. 5A).…”

Section: The Effects Of Mutations In the Effector Loop Region Of Harascontrasting

confidence: 93%

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Distinct Mechanisms of α5β1 Integrin Activation by Ha-Ras and R-Ras

Kinashi¹,

Katagiri²,

Watanabe³

et al. 2000

Journal of Biological Chemistry

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To investigate the possible roles of the Ras/Rho family members in the inside-out signals to activate integrins, we examined the ability of Ras/Rho small GTPases to stimulate avidity of ␣ 5 ␤ 1 (VLA-5) to fibronectin in bone marrow-derived mast cells. We found that both HaRas Val-12 activated VLA-5 through PI 3-kinase p110␦. The mutational effects of the R-Ras effector loop region on adhesion were not correlated with PI 3-kinase activities, consistent with our contention that R-Ras has a distinct pathway to modulate avidity of VLA-5.Adhesion mediated by integrins controls cell migration and localization. Adhesive interactions through integrins are modulated by adhesiveness (avidity) as well as expressions of integrins (1, 2). Although both types of regulations are important, avidity modulation of integrins in particular plays a critical role in leukocyte migration and localization during inflammatory responses (3). Several external stimuli were reported to modulate avidity of integrins without changes of integrin expressions, such as antigen, chemokines, and cytokines (3-5). A rapid change of avidity of integrins occurs within minutes and is triggered by intracellular signaling pathways, which are referred to as inside-out signals (6).Avidity modulation of integrins is regulated by increasing affinity to ligands, or by spatial redistribution of integrins on cell surface, which increases the number of integrins on the contact site (7-10). Which types of avidity regulations are utilized largely depends on stimuli that induce adhesion. PMA 1 enhanced adhesion without detectable change in ligand-binding affinity of integrins (11-13). Recent studies have shown that an increase in lateral diffusion and clustering of integrins by PMA or cytochalasin D at low doses facilitates adhesion (10,14), suggesting that the adhesion is mediated by low affinity, but multivalent bindings of integrins. On the other hand, affinity modulation in integrins detected with soluble ligands or antibodies recognizing the high affinity state was reported for ␣ 4 ␤ 1 , ␣ 5 ␤ 1 , ␣ L ␤ 2 , and ␣ IIb ␤ 3 integrins in cells stimulated with activating antibodies, manganese ions, or cross-linking of the T cell receptor (12,13,(15)(16)(17)(18)(19). We previously demonstrated that PMA-stimulated mast cells adhered to fibronectin without accompanying affinity modulation of VLA-5, while Fc⑀RI crosslinked mast cells adhered to fibronectin by the high affinity state of VLA-5. Steel factor-induced adhesion was considered to be brought by both mechanisms (20). Changes in the affinity state of integrins influenced cell migratory speeds on substrates (21). However, the physiological significance of two modes of avidity modulation has not yet been demonstrated clearly.The Ras/Rho family of small GTPases regulates the actin cytoskeleton and contributes to the formation of membrane ruffling and focal adhesion (22,23). Cytoskeletal reorganization subsequent to attachment to substrate leads to marked cell shape changes and strengthens adhesive interactions. Sever...

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Section: The Effects Of Mutations In the Effector Loop Region Of Harascontrasting

confidence: 93%

“…The 80-kDa fibronectin fragment was radioiodinated with a modified method using chloramine T (37). The typical specific activity of the labeled 80-kDa fragment used in our experiments was about 3.5 ϫ 10 8 dpm/nmol.…”

Section: Methodsmentioning

confidence: 97%

Distinct Mechanisms of α5β1 Integrin Activation by Ha-Ras and R-Ras

Kinashi¹,

Katagiri²,

Watanabe³

et al. 2000

Journal of Biological Chemistry

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“…AGE-BSA was radiolabelled with [ 125 I] using chloramine-T as described [21]. To examine if anti-p60 antibody can inhibit the binding of [ 125 I] AGE-BSA cells were grown to near confluency in 24-well plates.…”

Section: Preparation Of Age-modified Bovine Serum Albumin (Bsa)mentioning

confidence: 99%

Toxic action of advanced glycation end products on cultured retinal capillary pericytes and endothelial cells: relevance to diabetic retinopathy

et al. 1997

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Summary The toxic effects of advanced glycation end products (AGEs) on bovine retinal capillary pericytes (BRP) and endothelial cells (BREC) were studied. AGE-modified bovine serum albumin (AGE-BSA) was toxic to BRP. At a concentration of 500 m g/ml it reduced the BRP number to 48 ± 3 % (p < 0.05) of untreated controls, as determined by cell counting with haemocytometer. AGE-BSA was also toxic to bovine aortic endothelial cells (BAEC) reducing cell number to 84 ± 3.1 % of untreated controls. Under similar conditions, low concentrations (62.5 m g/ml) of AGE-BSA were mitogenic to BREC increasing the cell proliferation to 156 ± 11 % (p < 0.05) above that of untreated controls. At a higher dose of 500 m g/ml AGE-BSA decreased the proliferation of BREC to 85 ± 6 % of untreated controls. Immunoblot analysis demonstrated that BRP and BREC express the p60 AGE-receptor. Retinal capillary bed from the human also stained positively for the p60 AGE-receptor. Addition of 0.25 m g/ml of p60 AGE-receptor antibody was able to block the effects of AGE-BSA on BRP and BREC. The level of binding of [ 125 I]-labelled AGE-BSA to the cell surface was small but significant among the three cell types. There was also an increase in the internalized pool of radioligand in BRP and BREC but this was very much lower than in BAEC. In all the cell types the internalized pool of [ 125 I]-labelled AGE-BSA was much larger than the amount associated with the cell surface. Degradation products were not detected in the media over the 24-h incubation of the cells with [ 125 I]AGE-BSA. The binding of [ 125 I]-labelled AGE-BSA to the cell surface was prevented by the addition of p60 AGE-receptor. These results suggest that the interaction of AGE-modified proteins with the membrane-bound AGE-receptor may play an important role in the pathogenesis of diabetic retinopathy. [Diabetologia (1997) 40: 156-164]

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“…Recombinant BPSV GIF was partially purified, from CHO cell-free supernates, by Mono-Q anionexchange chromatography followed by gel filtration on Superdex 200 (GE Healthcare Life Sciences). For ligand blotting, the purified GIF proteins were radio-iodinated by the chloramine-T method (McConahey & Dixon, 1980) and further purified on Superdex 200 columns in PBS with 0.04 % (v/v) CHAPS buffer, pH 7.2.…”

Section: Methodsmentioning

confidence: 99%

Conservation and variation of the parapoxvirus GM-CSF-inhibitory factor (GIF) proteins

Deane

Ueda

Wise

et al. 2009

Journal of General Virology

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The GIF protein of orf virus (ORFV) binds and inhibits the ovine cytokines granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). An equivalent protein has so far not been found in any of the other poxvirus genera and we therefore investigated whether it was conserved in the parapoxviruses. The corresponding genes from both the bovine-specific pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) were cloned and sequenced. The predicted amino acid sequences of the PCPV and BPSV proteins shared 88 and 37 % identity, respectively, with the ORFV protein. Both retained the six cysteine residues and the WSXWS-like motif that are required for biological activity of the ORFV protein.However, an analysis of the biological activity of the two recombinant proteins revealed that, whilst the PCPV GIF protein bound to both ovine and bovine GM-CSF and IL-2 with very similar binding affinities to the ORFV GIF protein, no GM-CSF-or IL-2-binding activity was found for the BPSV protein.

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[11] Radioiodination of proteins by the use of the chloramine-T method

Cited by 219 publications

References 6 publications

Distinct Mechanisms of α5β1 Integrin Activation by Ha-Ras and R-Ras

Distinct Mechanisms of α5β1 Integrin Activation by Ha-Ras and R-Ras

Toxic action of advanced glycation end products on cultured retinal capillary pericytes and endothelial cells: relevance to diabetic retinopathy

Conservation and variation of the parapoxvirus GM-CSF-inhibitory factor (GIF) proteins

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