Kinase receptor activation (KIRA): a rapid and accurate alternative to end-point bioassays

Sadick, Michael D.; Intintoli, Anthony; Quarmby, Valerie; McCoy, April; Canova-Davis, Eleanor; Ling, Victor

doi:10.1016/s0731-7085(98)00144-7

Cited by 59 publications

(47 citation statements)

References 19 publications

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“…Based on previously published data by Fukumoto et al we speculate the positive effect seen on total cartilage production by insulin is likely mediated through the IGF-1 pathway [14]. Fukumoto et al demonstrated [55]. They demonstrated that while insulin binds the IGF-1 receptor directly, the activity of insulin in the IGF-1 KIRA is more than 100-fold less than the IGF-1 ligand.…”

Section: Treatmentmentioning

confidence: 88%

Serum‐free media for periosteal chondrogenesis in vitro

Fitzsimmons

Sanyal

Gonzalez

et al. 2004

Journal Orthopaedic Research

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Organ culture studies involving whole explants of periosteum have been useful for studying chondrogenesis, but to date the standard culture model for these explants has required the addition of fetal bovine serum to the media. Numerous investigators have succeeded in culturing chondrocytes and embryonic cells in serum-free conditions but there have been no studies focused on achieving a defined, serum-free media for culturing periosteal explants. The purpose of the present investigation was to determine if whole periosteal explants can be grown and produce cartilage in serum-free conditions, and to define the minimum media supplements that would be conducive to chondrogenesis. 321 periosteal explants were obtained from the medial proximal tibiae of 31 two month-old NZ white rabbits and cultured using a published agarose suspension organ culture model and DMEM for six weeks. The explants were cultured with and without fetal bovine serum or bovine serum albumin and exposed to transforming growth factor beta alone, a combination of growth factors we call ChondroMix (10 ng/ml transforming growth factor beta, 50 ng/ml basic fibroblast growth factor, and 5 pg/ml growth hormone), and/or ITS+ (2.08 pg/ml each of insulin, transferrin, and selenious acid, plus 1.78 pg/ml linoleic acid and 0.42 mg/ml BSA). Maximal chondrogenic stimulation in this study was observed with the combination of ChondroMix and ITS+. However, the minimal requirement to match or exceed the level of chondrogenic stimulation seen in the standard model (TGF-1 in 100/0 FBS) was achieved simply by the addition of 2.0 pg/ml insulin in 0.10/0 BSA-containing medium (p < 0.05). Therefore, based on our results, it would be reasonable to assume that insulin is the component in ITS+ responsible for the observed increase in total cartilage growth. Lower concentrations of insulin were not effective, suggesting that the observed effect of insulin requires activation of the IGF-1 receptor.

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Section: Treatmentmentioning

confidence: 88%

Serum‐free media for periosteal chondrogenesis in vitro

Fitzsimmons

Sanyal

Gonzalez

et al. 2004

Journal Orthopaedic Research

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“…Therefore, modernb ioassays detectingt he initial commitment of ac ell to respond (i.e. intracellular signals) following the ligand activation have been developed (40,41).T he IGF-I receptor belongs to the tyrosinek inase receptor superfamily ( 42,43).T he auto-phosphorylation of tyrosine residues aftert he stimulation of IGF-I receptor (ligand binding) is the first step of the intracellular signal cascade. Accordingly, we have recentlyd eveloped a highlys pecificI GF-I KIRA, whichh as enabled us to determine thea bility of serum to activate (i.e.…”

Section: Discussionmentioning

confidence: 99%

Changes in bioactive IGF-I and IGF-binding protein-1 during an oral glucose tolerance test in patients with liver cirrhosis

et al. 2006

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Objective :L iver cirrhosis is characterized by reduced circulating IGF-I and this has been linked to an adverse clinical outcome.Therefore, we investigated the dynamic changes in circulating total, free, and bioactiveI GF-I, IGF-binding protein (IGFBP)-1, IGFBP-2, and IGFBP-1-bound IGF-I (binarycomplex) during an oral glucose tolerance test (OGTT) in patients with liver cirrhosis. Methods:Seven Caucasian males with liver cirrhosis and seven healthymales matched for age (54.4G 3.2 vs 54.6G 4.4 years) and body mass index( 25.3G 1.2 vs 25.9G 1.3 kg/m 2 )w ere studied. Blood samples were drawn at 0, 30, 60, 90, 120, 150, and 180 min for determination of serum total and free IGF-I, IGFBP-1, IGFBP-2, and binarycomplex, while bioactiveIGF-I wasmeasured at 0, 30, 60, 120, and 180 min. Results:Incomparison with healthysubjects, baseline levels of total (47%), free (36%), and bioactive IGF-I (51%) were lower,w hile IGFBP-1 (268%) wash igher ( P ! 0.05), IGFBP-2 (172%) tended to be higher ( P O 0.05), and the binarycomplexunchanged ( w 100%) in cirrhotic patients. Serum total and free IGF-I, and IGFBP-2 remained unchangedinb oth study groups during the OGTT.B ioactiveIGF-I decreased by 29% from baseline to 60 min in cirrhotic patients and remained low at the end of the OGTT ( P ! 0.05). As imilar tendency waso bservedi nh ealthyc ontrols ( P Z 0.052). Concomitantly, IGFBP-1, binarycomplex, and IGFBP-1 saturation indexd ecreased significantlyi nb oth groups. The disappearance of the binarycomplex wasa bout twofold faster than that of IGFBP-1 ( P ! 0.05). Conclusion:D espite unchangedc oncentrations of total and free IGF-I, bioactive IGF-I declined significantlya fter an oral glucose load in patients with liverc irrhosis and the same tendency was observedinhealthysubjects. We speculate that the reduction in bioactiveIGF-I mayberelated to the higher levels of free IGFBP-1 and the faster disappearance of IGFBP-1-bound IGF-I. 155 285-292 European Journal of Endocrinology

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“…Fractions containing the eluted IGF-1-His were pooled, dialyzed, aliquotted, and kept at Ϫ 80C until used. The biological activity of the IGF-1-His chimeric protein was identical to that of the native IGF-1, as evaluated by its potential to induce the phosphorylation of its receptor in a KIRA assay (Sadick et al 1999).…”

Section: Methodsmentioning

confidence: 99%

Novel Non-isotopic Method for the Localization of Receptors in Tissue Sections

Desnoyers¹,

Simonette²,

Vandlen³

et al. 2001

J Histochem Cytochem.

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S U M M A R YWe describe a novel fluorescent method for the detection of receptors for chimeric proteins in tissue sections. The technique was developed using a recombinant human insulin-like growth factor (IGF-1) chimera, bearing six additional histidine residues at the carboxy-terminal end (IGF-1-His). We demonstrated that dehydration of the tissue sections was detrimental for binding and that its prevention dramatically increased sensitivity. The specificity of IGF-1-His interaction was shown by gradual abolition of the fluorescent signal in the presence of increasing concentrations of IGF-1. Combining immunofluorescence with in situ ligand binding, we showed that IGF-1-His binding corresponded to the IGF-1 receptor (IGFR-1) distribution in human fetal kidney. Moreover, incubation of the tissue sections with an anti-IGFR-1 blocking antibody abolished IGF-1-His binding, demonstrating that the interaction was mediated by the IGFR-1. The method was also used to localize the IGFR-1 in E18 rat embryo sagittal sections. The IGF-1-His binding pattern was observed in brain, cartilage, lung, skin, heart, diaphragm, and tongue, and paralleled the previously reported IGFR-1 distribution. We believe that this new non-isotopic in situ ligand binding method will facilitate rapid and accurate localization of receptors in tissue sections.

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Kinase receptor activation (KIRA): a rapid and accurate alternative to end-point bioassays

Cited by 59 publications

References 19 publications

Serum‐free media for periosteal chondrogenesis in vitro

Serum‐free media for periosteal chondrogenesis in vitro

Changes in bioactive IGF-I and IGF-binding protein-1 during an oral glucose tolerance test in patients with liver cirrhosis

Novel Non-isotopic Method for the Localization of Receptors in Tissue Sections

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