Esta: A Bioassay System for the Determination of the Potencies of Hormones and Antibodies Which Mimic Their Action.

Ealey, P. A.; Yateman, M; Holt, S. J.; Marshall, Nicholas J.

doi:10.1677/jme.0.001r001

Cited by 49 publications

(21 citation statements)

References 7 publications

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“…Thus, exposure of small pieces of mouse epidermis, trachea, esophagus, tongue, liver, and cardiac muscle to LCM-EGTA for up to 6 h did not disrupt desmosome adhesion, as shown by transmission electron microscopy, even though separation of the cell membranes and vacuolation of cells indicated that the tissues were clearly affected by calcium removal (Figure 3). Parallel immunofluorescence studies indicated that E-cadherin in tissues was internalized during the exposure to LCM-EGTA (our unpublished results), and 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide assay (Ealey et al, 1988) showed that the tissues remained viable after this treatment.…”

Section: The Calcium Dependence Of Desmosomes Changes With Time In Cementioning

confidence: 54%

The α Isoform of Protein Kinase C Is Involved in Signaling the Response of Desmosomes to Wounding in Cultured Epithelial Cells

Wallis

Lloyd

Wise

et al. 2000

MBoC

158

211

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Initiation of reepithelialization upon wounding is still poorly understood. To enhance this understanding, we focus here on changes in the adhesive state of desmosomes of cultured Madin-Darby canine kidney cells in response to wounding of confluent cell sheets. Previous results show that desmosomal adhesion in Madin-Darby canine kidney cells changes from a calcium-dependent state to calcium independence in confluent cell sheets. We show that this change, which requires culture confluence to develop, is rapidly reversed upon wounding of confluent cell sheets. Moreover, the change to calcium dependence in wound edge cells is propagated to cells hundreds of micrometers away from the wound edge. Rapid transition from calcium independence to calcium dependence also occurs when cells are treated with phorbol esters that activate PKC. PKC inhibitors, including the conventional isoform inhibitor Gö 6976, cause rapid transition from calcium dependence to calcium independence, even in subconfluent cells. The cellular location of the ␣ isoform of PKC correlates with the calcium dependence of desmosomes. Upon monolayer wounding, PKC␣ translocates rapidly to the cell periphery, becomes Triton X-100 insoluble, and also becomes concentrated in lamellipodia. The PKC␣ translocation upon wounding precedes both the increase in PKC activity in the membrane fraction and the reversion of desmosomes to calcium dependence. Specific depletion of PKC␣ with an antisense oligonucleotide increases the number of cells with calcium-independent desmosomes. These results show that PKC␣ participates in a novel signaling pathway that modulates desmosomal adhesion in response to wounding.

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Section: The Calcium Dependence Of Desmosomes Changes With Time In Cementioning

confidence: 54%

The α Isoform of Protein Kinase C Is Involved in Signaling the Response of Desmosomes to Wounding in Cultured Epithelial Cells

Wallis

Lloyd

Wise

et al. 2000

MBoC

158

211

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“…The potency of the hormone tion, [12][13][14] local release of this hormone from hormonewas measured with a uniquely precise bioassay which loaded PMMA may promote early postoperative bone is based upon the eluted stain assay system (ESTA) which we recently developed. 18,19 The immunoactivity of the hGH released was also monitored using a con-duced by the incorporation into the methacrylate cedisc weight was generally 5-6%. These weights were used to calculate the weight-corrected percentage rement.…”

Section: Introductionmentioning

confidence: 99%

“…21 They were routinely grown in suspension culThis was followed by careful mixing (10 min) to ensure ture as described previously. 18,19 homogeneity, and finally, the addition of the liquid monomer. The cements were allowed to dough and Bioassay were then cast into discs in PTFE molds (10 mm in diameter and 7 mm deep).…”

Section: Introductionmentioning

confidence: 99%

“…The cements were allowed to dough and Bioassay were then cast into discs in PTFE molds (10 mm in diameter and 7 mm deep). Immediately after polymerFor the bioassays the cells were prepared as described previously 18,19 and dispensed into microtiter ization the discs were weighed; the variability in plates (50 ȐL), at a density of 8 ϫ 10 5 cells/mL. This whereas between-assay imprecision was 3.5, 5.2, and 5.5% at hGH concentrations of 7.7, 21.7, and 45.8 mU/ was followed by addition of the appropriate standard or sample (50 ȐL), which was also diluted in bioas-L, respectively.…”

Section: Introductionmentioning

confidence: 99%

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Investigation into the release of bioactive recombinant human growth hormone from normal and low-viscosity poly(methylmethacrylate) bone cements

Goodwin

Braden

Downes

et al. 1997

J. Biomed. Mater. Res.

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Previous studies showed that recombinant human growth hormone (hGH) released from hormone-loaded poly(methylmethacrylate) (PMMA) cement stimulated osteoid formation in a rabbit model. Local delivery of hGH from cemented hip arthroplasties may thereby provide a means of reducing the problem of aseptic loosening. We have investigated two different formulations of PMMA as delivery systems for bioactive hGH. The bioactivity of the hormone release in vitro was monitored with an eluted stain assay (ESTA). The hGH was also measured by an immunoassay, which provides an alternative assessment of structural integrity of the hormone released. In addition, we adapted the ESTA bioassay to assess the in vitro cytotoxicity of the cements. Using unloaded cements, the undiluted eluates from both types of PMMA proved cytotoxic. This cytotoxicity could be diluted out, and the procedure allowed us to measure the bioactivity of hGH in the eluates from hormone-loaded cements independent of their cytotoxicity. The major fraction of the bioactivity was released from both of the PMMA cements during the first 24 h, but the hormone remained detectable in eluates collected after 36 days of elution. Comparison of the bio- and immunoactivity of the hGH released showed that the ratio of these two activities (i.e., the B:I ratio) was constant over this time period. However in parallel studies in which hormone-loaded discs were stored under dry conditions prior to elution, we found that the B:I ratio then declined markedly. This suggests that fully hydrated conditions, such as when the discs are bathed in assay medium, are necessary to maintain the bioactivity of the hGH. Both cements released only approximately 1% of the hormone originally incorporated, but the hGH concentration which accumulated in the eluates were high in physiologic terms (approximately 1000 mU/L).

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“…The concentrations of bioactive hGH were determined using the ESTA bioassav. These detailed studies were possible because of the performance charac teristics of the ESTA bioassay, which has the precision and high sample capacity which is normally only attain able with immuno-or radioligand assay [1][2][3][4][5][6]. The ESTA bioassay is based upon the response of Nb2 rat lymphoma cells to lactogens such as hGH.…”

Section: Introductionmentioning

confidence: 99%

An Investigation into the Lability of the Bioactivity of Human Growth Hormone Using the ESTA Bioassay

et al. 1996

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We compared the bioactivity attributable to human growth hormone (hGH) in serum samples, determined at the time of their collection, with that after storage for 2-18 months at -20 °C. The samples were obtained from volunteers and patients who underwent provocative tests of hGH secretion, and the bioactivity was determined in the ESTA bioassay, which is based upon the use of Nb2 cells. We report that, in some subjects, the bioactivity of samples collected at the response peaks deteriorated on storage for as little as 2 months. The decrease in hGH bioactivity was systematic in that it consistently declined so as to approach the values initially determined by an immunoassay (Hybritech IRMA). This differential lability was a characteristic of the peak samples, and was not observed for either samples collected before and after the peaks of hGH secretion or for purified preparations of hGH which were subjected to a range of freeze/thaw and storage regimens. We suggest that this unusual lability is indicative of transient shifts in the spectrum of the variants of hGH which are present in the circulation following stimulation by provocative agents. This study emphasises the need to minimise the risk of introducing storage artefacts in investigations into the responses of hGH to provocation.

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Esta: A Bioassay System for the Determination of the Potencies of Hormones and Antibodies Which Mimic Their Action.

Cited by 49 publications

References 7 publications

The α Isoform of Protein Kinase C Is Involved in Signaling the Response of Desmosomes to Wounding in Cultured Epithelial Cells

The α Isoform of Protein Kinase C Is Involved in Signaling the Response of Desmosomes to Wounding in Cultured Epithelial Cells

Investigation into the release of bioactive recombinant human growth hormone from normal and low-viscosity poly(methylmethacrylate) bone cements

An Investigation into the Lability of the Bioactivity of Human Growth Hormone Using the ESTA Bioassay

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