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Biomedical subjects

E C Nice

Publications and source records attributed to E C Nice.

At least 73 records · Page 4Linked to original sources
PubMed1986

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry.

Carcinoma, Squamous Cell↗
PubMed1986

In vitro actions on hemopoietic cells of recombinant murine GM-CSF purified after production in Escherichia coli: comparison with purified native GM-CSF.

Recombinant murine GM-CSF produced in Escherichia coli was purified to homogeneity and tested in parallel with purified native GM-CSF. Both recombinant and native GM-CSF stimulated granulocyte and/or macrophage colony formation by adult and fetal mouse progenitor cells, and with adult marrow cells the specific activity of the recombinant GM-CSF (25 X 10(8) U/mg) was similar to that of the native form (15 X 10(8) U/mg). At high concentrations (greater than 200 U/ml), both forms of GM-CSF also stimulated eosinophil colony formation by adult marrow cells and, at very high concentrations (greater than 800 U/ml), megakaryocyte and some erythroid and mixed-erythroid colony formation. Recombinant GM-CSF was as effective in stimulating the proliferation of the GM-CSF-dependent cell line FD as the native molecule. Both recombinant and native GM-CSF were able to induce partial differentiation in colonies of WEHI-3B myeloid leukemic cells. Recombinant GM-CSF competed effectively for the binding of 125I-labeled native GM-CSF to hemopoietic cells, and antiserum to recombinant GM-CSF also neutralized the biological activity of native GM-CSF. The bacterially synthesized GM-CSF was a slightly more effective stimulus for megakaryocyte colony formation than the native molecule. The demonstration that purified bacterially synthesized GM-CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.

Animals↗
PubMed1986

Identification of two anti-parallel beta-sheet conformations in the solution structure of murine epidermal growth factor by proton magnetic resonance.

Epidermal growth factor (EGF) is a small mitogenic protein. Proteins with sequence homology with EGF or with its membrane-bound protein receptor have been proposed to play a role in oncogenesis. This report describes solution NMR data that provide evidence that the solution conformation of murine EGF includes an anti-parallel beta-sheet structure involving residues S2-P4, V19-I23, and S28-N32; a small anti-parallel beta-sheet involving residues Y37-S38 and T44-R45; and a multiple-bend (or short irregular helix) structure for residues C6-C14 that is disulfide bonded to the V19-I23/S28-N32 beta-sheet. Implications of these results for structure and function studies of EGF and for molecular design of EGF and homologous alpha-type transforming growth factors are discussed.

Animals↗
PubMed1986

Use of microbore high-performance liquid chromatography for purifying subnanomole levels of polypeptides for microsequencing. Structural studies on the murine plasma cell antigen PC-1.

A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed-phase high performance liquid chromatography on short (10 cm or less) microbore (1-2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC-1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed-phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (greater than 90%) and in small eluent volumes (40-60 microL) which can be loaded directly onto the gas-phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode-array detector for identifying tryptophan-containing peptides from on-the-fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan-containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.

Amino Acid Sequence↗
PubMed1985

Rat epidermal growth factor: complete amino acid sequence. Homology with the corresponding murine and human proteins; isolation of a form truncated at both ends with full in vitro biological activity.

Epidermal growth factor (EGF) isolated from the submaxillary gland of the rat (rEGF) is missing the COOH-terminal five residues present in both mouse and human EGF. rEGF competes for the binding of 125I-labelled mEGF to human carcinoma cells with the same affinity as mEGF. rEGF and mEGF have identical mitogenic activities on mouse 3T3 fibroblasts, thus the C-terminal region of the sequence is not necessary for the in vitro activity of EGF. Using reversed-phase high-performance liquid chromatography, four molecular forms of EGF have been extracted from rat submaxillary glands. These forms represent rEGF, rEGF(2-48), rEGF(3-48) and rEGF(4-48); all forms appear to be equipotent in both the receptor binding and mitogenic assays. The isoelectric points of these rEGFs are in the range of pH 5.1 to 5.2. The primary structure of rEGF was determined from approximately 10 micrograms protein by sequence analysis of the intact molecule and fragments obtained from the reduced and alkylated protein by chemical cleavage with CNBr and enzymic cleavage with chymotrypsin and a proline-specific endopeptidase. Subnanomole amounts of generated peptides were purified to homogeneity by reversed-phase microbore high-performance liquid chromatography and analysed by automated Edman degradation in a gas-phase sequencer. There are 48 amino acid residues in the complete polypeptide chain which lacks alanine, phenylalanine, lysine and tryptophan. The amino acid sequence of rat epidermal growth factor is: Asn-Ser-Asn-Thr-Gly-Cys-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-Cys-Leu-Asn- Gly-Gly-Val-Cys-Met-Tyr-Val-Glu-Ser-Val-Asp-Arg-Tyr-Val-Cys-Asn-Cys -Val-Ile-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Gln-His-Arg-Asp-Leu-Arg. The calculated relative molecular mass from the sequence analysis is 5377.

Amino Acid Sequence↗
PubMed1985

Purification of two forms of colony-stimulating factor from mouse L-cell-conditioned medium.

A modified procedure for the purification of the colony-stimulating factors (CSFs) in mouse L-cell-conditioned medium is used to isolate two forms of CSF, which are separable by reversed-phase high performance liquid chromatography with 300-A pore size supports. The specific biological activity of these CSFs (2 X 10(9) colonies/mg) was considerably higher than has been achieved by other methods. Even at high concentration (200 pM) both molecules stimulated predominantly more macrophage than granulocyte colonies; however, the less hydrophobic form appeared to stimulate the formation of more pure granulocytic colonies. Almost twice as much of the less hydrophobic CSF was recovered from L-cell-conditioned medium. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both forms of L-cell CSF had apparent molecular masses of approximately 70,000 daltons. However, on reduction with 2-mercaptoethanol, while both forms generated a 39,000-dalton subunit, the less hydrophobic form also yielded a 32,000-dalton subunit. Storage of either form of L-cell CSF at pH 2.1, in the presence of acetonitrile or isopropanol, destroyed the biological activity. Electrophoretic analysis of the L-cell CSFs stored under these conditions indicated that this was associated with a spontaneous dissociation of the CSF dimer into the inactive subunits. There was some charge heterogeneity (pI 3.5-4.7) indicating different degrees of glycosylation. The unique N-terminal amino acid sequences of both forms of CSF were the same: (Lys-Glu-Val-Ser-Glu-His-X-Ser-His-Met-Ile-Gly-Asn). Thus, the polypeptide chains appear to be identical for the subunits of both forms of L-cell CSF.

Amino Acids↗
PubMed1985

A microbore high-performance liquid chromatography strategy for the purification of polypeptides for gas-phase sequence analysis. Structural studies on the murine transferrin receptor.

We describe herein the use of reversed-phase high-performance liquid chromatography coupled with the novel application of short (10 cm or less) microbore columns (2 mm internal diameter) to fractionate and purify a number of tryptic peptides generated from approximately 200 pmol purified murine transferrin receptor. The use of reversed-phase microbore columns permits the recovery of submicrogram amounts of purified polypeptides in high yield (greater than 90%) in small eluent volumes (20-60 microliter). In this manner, purified polypeptides can be loaded directly onto the gas-phase sequencer without further manipulation. This procedure avoids sample loss, which frequently occurs with other forms of concentration (e.g. lyophilization, evaporation). The application of second-order-derivative ultraviolet spectroscopy, using a diode array detector, for the analysis of aromatic aminoacid-containing peptides in complex tryptic digests is described. N-terminal amino acid sequence analyses were performed on six tryptic peptides, yielding 105 unique assignments; this corresponds to approximately 14% of the molecule. A comparison of this amino acid sequence information with the primary structure of human transferrin receptor deduced from the mRNA sequence [Nature (Lond.) 311, 675-678 (1984); Cell 39, 267-274 (1984)] reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.

Amino Acid Sequence↗
PubMed1984

In situ cyanogen bromide cleavage of N-terminally blocked proteins in a gas-phase sequencer.

Herein we describe a procedure for the in situ cyanogen bromide cleavage of N-terminally blocked proteins which have been immobilised onto the glass fiber sample disk of the gas-phase sequencer. In this manner, new amino terminii suitable for automated Edman degradation can be generated. Cytochrome C was cleaved using this method on the carboxyl side of methionine residues 65 and 80. This allowed sequence analysis to begin simultaneously at residues 66 and 81 (Table 1). This procedure offers an alternative sequencing tactic for methionine-containing proteins which are N-terminally blocked.

Amino Acid Sequence↗
PubMed1983

High-performance liquid chromatographic methods for the analysis of human parathyroid hormone in reference standards, parathyroid tissue and biological fluids.

Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to fractionate human parathyroid hormone (hPTH) from a variety of natural sources and to compare it with synthetic hPTH and hPTH fragments. Multiple radioimmunoassay systems for amino, mid and carboxyl regions of hPTH were used to monitor various preparations of hPTH previously prepared by conventional methods and ampouled in nanogram amounts for reference standard and reagent purposes. Results confirmed that they were free of detectable cleavage products, but showed that the intact hPTH comprised three or four closely associated components. A similar pattern of heterogeneity was obtained when hPTH was extracted from stored human parathyroid adenomata by a simple rapid HPLC bulk fractionation method. Comparison with synthetic 1-84 hPTH and modification of sample handling to minimize oxidative conditions, indicate that some of these components are probably intermediate oxidation products. A number of less hydrophobic components, with carboxyl region immunoreactivities, were obtained from the individual adenoma samples, human parathyroid cyst fluid, ampouled samples of human adenoma tissue culture medium, and secondary hyperparathyroid plasma ultrafiltrate when they were fractionated by RP-HPLC. The results strongly suggest that the biological degradation of hPTH is more complex than generally believed, and that RP-HPLC offers a new dimension in its analysis.

Adenoma↗
PubMed1983

A synthetic peptide containing the autophosphorylation site of the transforming protein of Harvey sarcoma virus is phosphorylated by the EGF-stimulated tyrosine kinase.

The transforming proteins (p21) of Harvey and Kirsten sarcoma viruses threonine kinase activity, which phosphorylates threonine 59 of the p21 proteins themselves. A tridecapeptide: Arg-Arg-Leu56-Asp-Thr-Thr59-Gly-Gln-Glu-Tyr-Ser-Ala66 containing residues 56-66 of p21 is phosphorylated solely on tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Km-Values of 240 and 80 microM and Vmax values of 1.7 and 0.1 nmol.min-1.mg-1 were obtained in the presence and absence of EGF, respectively.

Carcinoma, Squamous Cell↗
PubMed1983

Murine epidermal growth factor: heterogeneity on high resolution ion-exchange chromatography.

We have shown that epidermal growth factor (EGF) purified either by the classical method of Savage and Cohen, or solely by h.p.l.c. techniques can be resolved into two species, EGF alpha and EGF beta. However, despite the apparent purity of such materials, as determined both chromatographically and by amino acid analysis, they failed to give homogeneous products on radioiodination. Analysis by isoelectric focusing on agarose gels followed by transfer to nitrocellulose and silver staining showed that EGF alpha could be further resolved into three sub-species which focused at pH 4.6, 4.3 and 4.1. EGF beta (which also focused at pH 4.6) contained very small amounts of the species with isoelectric points of 4.1 and 4.3, probably due to slight contamination of this preparation by EGF alpha. Preparative separation of the sub-species of EGF alpha was achieved by high performance anion-exchange chromatography at pH 6.5 on a Pharmacia Mono Q column. Radioiodination of these purified sub-species did not produce significant charge heterogeneity. However, two slightly different forms of [125I]EGF alpha 1 (pH 4.6 species) were separable by anion-exchange chromatography on the Mono Q column. All of the EGF species competed for binding to EGF receptors on A431 cells and were active mitogens for BALB/c 3T3 fibroblasts.

Animals↗
PubMed1983

Differences in the Elution profiles of corticotropin-releasing activity in rat plasma and hypothalamic extracts following high performance liquid chromatography.

Extracts of rat plasma and hypothalami were fractionated by reversed-phase high performance liquid chromatography (HPLC) and the fractions were assayed in an in vitro bioassay for corticotropin-releasing activity. Corticotropin releasing factor (CRF) bioactivity was found in many fractions with retention times of 20-50 min. In contrast, material derived from the plasma of rats stressed by ether anaesthesia yielded only one peak of CRF bioactivity, with a retention time of 47-48 min. These results suggest that ACTH can be released from isolated pituitary cells by a number of components derived from HPLC of extracts of rat hypothalami. It is possible, however, that some of these are artefacts of the extraction process from post-mortem material. Because the CRF activity found in plasma was homogeneous by HPLC, plasma can be used to obtain physiologically active CRF.

Animals↗
PubMed1982

Polymorphically acetylated aminoglutethimide in humans.

The urinary excretion during 24 h of aminoglutethimide (AG) its major metabolite (N-acetylAG) and two minor metabolites (N-formylAG and nitroG) were measured in 10 volunteers given AG who had been typed for acetylator phenotype using sulphadimidine. The slow acetylators of sulphadimidine excreted more AG (mean 28% of the administered dose) than did the fast acetylators (12%), but the latter excreted more of the dose as N-acetylAG (8.8%) than did the former (3.9%). NitroG and N-formylAG were minor urinary metabolites of AG in humans. The former was more abundant in the urine of slow acetylators (0.10% of the dose) than in that of fast acetylators (0.047%), whereas the respective proportions of doses excreted as the N-formyl derivative (0.475 and 0.465%) were not significantly different for the two acetylator phenotypes. These results show that AG is among those drugs that are polymorphically acetylated in humans.

Acetylation↗
PubMed1982

Two forms of murine epidermal growth factor: rapid separation by using reverse-phase HPLC.

Epidermal growth factor (EGF) has been isolated from acid extracts of C57BL6/J mouse submaxillary glands by using hydrophobic chromatography. High yields of EGF in large amounts (10 mg) can be isolated reliably from the acid extract of the glands in less than 4 hr. The reverse-phase HPLC techniques used to purify the EGF initially yielded what appeared to be a single homogeneous EGF molecule. However, ion pairing reagents (e.g., heptafluorobutyric acid) altered the chromatographic properties, revealing two distinct species: EGF-alpha and EGF-beta. The apparent molecular weights, isoelectric points, and antigenic properties of EGF-alpha and EGF-beta were identical, and both forms stimulated a mitogenic response in 3T3 cells. Analysis of different preparations of purified EGF (commercial and experimental) indicated the presence of EGF-alpha and EGF-beta in constant proportion. Previous EGF binding studies must have used mixtures of 125I-labeled EGF-alpha and 125I-labeled EGF-beta. The two molecules appear to compete for an identical receptor on the cell surface.

Animals↗
PubMed1982

Colony-forming cell proliferation: a rapid and sensitive assay system for murine granulocyte and macrophage colony-stimulating factors.

A microculture assay for murine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been developed using fetal liver GM colony-forming cells (CFC) isolated by fluorescence-activated cell sorting. These GM-CFC are free of mature hemopoietic cells, such as granulocytes and macrophages, which may interfere with direct assays for GM-CSF. The assay procedure allows the quantitation of GM-CSF within 48 hr by measuring the number of cells produced from 50 GM-CFC in microcultures (15 microliter). The assay is particularly simple to set up and score and yet, because of the reduced volumes, this assay is still capable of detecting 0.2 pg (i.e., 0.2 U) of GM-CSF within 48 hr, i.e., 100 times less GM-CSF than the conventional soft agar assay. By allowing the microcultures to develop for 7 days, the extra proliferation allows a further tenfold increase in the sensitivity of CSF detection. The time and cost of setting up hundreds of GM-CSF assays for fractions from chromatographic columns, e.g., reverse phase high performance liquid chromatography, is reduced by at least five-fold. Enough GM-CFC can be isolated and stored frozen in one afternoon to provide sufficient cells for the daily assay of 200 samples of GM-CSF for several months. Microassay results for several sources of GM-CSF at different stages of purification are compared to the results obtained from the soft agar assay.

Animals↗
PubMed1981

Purification and assay of bovine parathyroid hormone by reversed-phase high performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (HPLC) has been used to purify a crude extract of bovine parathyroid glands, in a single run on an analytical column, to give a high yield of homogeneous material with full bioactivity in in vivo bioassay. Bovine parathyroid hormone (bPTH) prepared and purified by conventional procedures has been rapidly and quantitatively separated from its oxidation and other degradation products, from hormone fragments and from non-hormonal contaminants. Recovery of bPTH, monitored by region-specific immunoassays, in vivo bioassay and re-chromatography on HPLC was greater than 93%. The detection limit of the HPLC system, using endogenous tryptophan fluorescence, was 20 ng bPTH.

Animals↗