Biomedical subjects

E C Nice

Publications and source records attributed to E C Nice.

At least 37 records · Page 2Linked to original sources

PubMed1999

Instrumental biosensors: new perspectives for the analysis of biomolecular interactions.

The use of instrumental biosensors in basic research to measure biomolecular interactions in real time is increasing exponentially. Applications include protein-protein, protein-peptide, DNA-protein, DNA-DNA, and lipid-protein interactions. Such techniques have been applied to, for example, antibody-antigen, receptor-ligand, signal transduction, and nuclear receptor studies. This review outlines the principles of two of the most commonly used instruments and highlights specific operating parameters that will assist in optimising experimental design, data generation, and analysis.

Animals↗

PubMed1998

Micro-sequencing strategies for the human A33 antigen, a novel surface glycoprotein of human gastrointestinal epithelium.

Monoclonal antibody (mAb) A33, which recognizes a M(r) approximately 43,000 differentiation antigen (A33) expressed in normal human colonic and small bowel epithelium as well as in 95% of colon cancers, shows specific targeting of colon cancer in humans and is currently being evaluated for clinical use. Here, we describe strategies for the purification and structural analysis of the A33 antigen from the human colorectal carcinoma cell lines LIM1215 and SW1222. Edman degradation of the intact protein and nine peptides, derived by proteolytic digestion of the A33 antigen with Asp-N endoproteinase, thermolysin, trypsin and pepsin followed by micropreparative reversed-phase high-performance liquid chromatography, allowed the unambiguous sequence assignment of 153 amino acid residues; these data reveal one N-glycosylation sequeon in Asp-N endoproteinase peptide D4, and a disulfide linkage between peptides D1 and D4. This amino acid sequence information has facilitated the cloning and subsequent sequencing of a cDNA for the A33 antigen which demonstrates that it is a novel human cell surface molecule of the immunoglobulin superfamily.

Amino Acid Sequence↗

PubMed1998

Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor.

Two synthetic analogues of murine epidermal growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF. The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF. Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF. [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its 1H chemical shifts suggested that its structure was also very similar to native.

Chromatography, High Pressure Liquid↗

PubMed1998

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization.

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti-ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M-1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.

Antibodies, Monoclonal↗

PubMed1997

Characterization of posttranslational modifications of human A33 antigen, a novel palmitoylated surface glycoprotein of human gastrointestinal epithelium.

Monoclonal antibody (mAb) A33 recognizes a differentiation antigen (A33) expressed in normal human gastrointestinal epithelium and in 95% of human colon cancers. Murine mAb A33 shows specific targeting of colon cancer in humans and a humanized A33 antibody is currently being evaluated in the clinic. The cDNA for the human A33 antigen has recently been cloned, and sequence comparison indicated that the A33 antigen is a novel human cell surface molecule of the immunoglobulin superfamily. Because mAb A33 recognizes a conformational epitope, only a partial characterization of the A33 antigen has been carried out to date. In this report we show that the A33 antigen is (I) N-glycosylated, containing approximately 8 K of N-linked carbohydrate and there is no evidence for O-glycosylation, sialylation or glycophosphatidylinositol, and (ii) S-acylated in vitro, incorporating [3H] palmitic acid linked through a hydroxylamine-sensitive thioester bond. The S-palmitoylation may be involved in regulating the internalization process initiated by binding of mAb A33 to cell surface A33 antigen.

Acylation↗

PubMed1997

Kinetic analysis of the interaction between the monoclonal antibody A33 and its colonic epithelial antigen by the use of an optical biosensor. A comparison of immobilisation strategies.

The interaction between the humanised A33 monoclonal antibody and the corresponding F(ab)'2 or Fab' fragments with the colonic epithelial A33 antigen, purified by micropreparative HPLC from membrane extracts of the colonic carcinoma cell line LIM 1215, has been studied with the BIAcore 2000 biosensor using surface plasmon resonance detection. The surface orientation of immobilised antibody and the Fab' fragment onto the biosensor surface was controlled using alternative immobilisation chemistries. This resulted in significantly higher molar binding activities compared with the conventional N-hydroxysuccinimide (NHS)/N-ethyl-N'-dimethylaminopropylcarbodiimide (EDC) chemistry. This increase in signal resulted in a concomitant increase in sensitivity of detection, which facilitates analysis of low levels of A33 antigen. The apparent association rate (ka) and dissociation rate (kd) constants obtained with the different immobilisation chemistries were determined. These analyses showed that the kinetic constants obtained for the IgG were not significantly affected by the method of immobilisation. F(ab)'2 and Fab' fragments immobilised using NHS/EDC chemistry showed significantly lower apparent affinity. By contrast the use of the thiol coupling chemistry with the Fab' fragment gave a five fold increase in observed KA, resulting in a similar affinity to that observed with the intact IgG molecule.

Antibodies, Monoclonal↗

PubMed1997

The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily.

The mAb A33 detects a membrane antigen that is expressed in normal human colonic and small bowel epithelium and > 95% of human colon cancers. It is absent from most other human tissues and tumor types. The murine A33 mAb has been shown to target colon cancer in clinical trials, and the therapeutic potential of a humanized antibody is currently being evaluated. Using detergent extracts of the human colon carcinoma cell lines LIM1215 and SW1222, in which the antigen is highly expressed, the molecule was purified, yielding a 43-kDa protein. The N-terminal sequence was determined and further internal peptide sequence obtained following enzymatic cleavage. Degenerate primers were used in PCRs to produce a probe to screen a LIM1215 cDNA library, yielding clones that enabled us to deduce the complete amino acid sequence of the A33 antigen and express the protein. The available data bases have been searched and reveal no overall sequence similarities with known proteins. Based on a hydrophilicity plot, the A33 protein has three distinct structural domains: an extracellular region of 213 amino acids (which, by sequence alignment of conserved residues, contains two putative immunoglobulin-like domains), a single hydrophobic transmembrane domain, and a highly polar intracellular tail containing four consecutive cysteine residues. These data indicate that the A33 antigen is a novel cell surface receptor or cell adhesion molecule in the immunoglobulin superfamily.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of a novel restricted antigen expressed by normal and transformed human colonic epithelium.

A cell surface antigen that is expressed by normal and 95% of transformed colonic epithelium and is recognized by the monoclonal antibody A33 (Welt, S., Divgi, C. R., Real, F. X., Yeh, S. D., Garin-Chesa, P., Finstad, C. L., Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., Carswell, E. A., Oettgen, H. F., and Old, L. J. (1990) J. Clin. Oncol. 8, 1894-1906) has been purified to homogeneity from the human colonic carcinoma cell line LIM1215. The A33 protein was purified from Triton X-114 extracts of LIM1215 cells under nondenaturing conditions. These extracts were applied sequentially to Green-Sepharose HE-4BD, Mono-Q HR 10/10, Superose 12 HR 10/30, and micropreparative Brownlee Aquapore RP 300. The purification was monitored by biosensor analysis using surface plasmon resonance detection with a F(ab')2 fragment of the humanized A33 monoclonal antibody immobilized on the sensor surface and Western blot analysis following SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions using humanized A33 monoclonal antibody. The purified A33 antigen has a Mr on SDS-PAGE of 43,000 under nonreducing conditions. By contrast, the purified protein displayed a Mr of approximately 180,000 under native conditions on both size exclusion chromatography and native PAGE, possibly due to the formation of a homotetramer. N-terminal amino acid sequence analysis of the purified protein identified 34 amino acid residues of a unique sequence: ISVETPQDVLRASQGKSVTLPXTYHTSXXXREGLIQWD. A polyclonal antibody was raised against a synthetic peptide corresponding to residues 2-20 of this sequence. The antipeptide serum recognized the purified protein using Western blot analysis under both nonreducing (Mr 43,000) and reducing (Mr 49,000) conditions.

Amino Acid Sequence↗

PubMed1996

Influence of interleukin-6 (IL-6) dimerization on formation of the high affinity hexameric IL-6.receptor complex.

The high affinity interleukin-6 (IL-6) signaling complex consists of IL-6 and two membrane-associated receptor components: a low affinity but specific IL-6 receptor and the affinity converter/signal transducing protein gp130. Monomeric (IL-6M) and dimeric (IL-6D) forms of Escherichia coli-derived human IL-6 and the extracellular ("soluble") portions of the IL-6 receptor (sIL-6R) and gp130 have been purified in order to investigate the effect of IL-6 dimerization on binding to the receptor complex. Although IL-6D has a higher binding affinity for immobilized sIL-6R, as determined by biosensor analysis employing surface plasmon resonance detection, IL-6M is more potent than IL-6D in a STAT3 phosphorylation assay. The difference in potency is significantly less pronounced when measured in the murine 7TD1 hybridoma growth factor assay and the human hepatoma HepG2 bioassay due to time-dependent dissociation at 37 degrees C of IL-6 dimers into active monomers. The increased binding affinity of IL-6D appears to be due to its ability to cross-link two sIL-6R molecules on the biosensor surface. Studies of the IL-6 ternary complex formation demonstrated that the reduced biological potency of IL-6D resulted from a decreased ability of the IL-6D (sIL-6R)2 complex to couple with the soluble portion of gp130. These data imply that IL-6-induced dimerization of sIL-6R is not the driving force in promoting formation of the hexameric (IL-6 IL-6R gp130)2 complex. A model is presented whereby the trimeric complex of IL-6R, gp130, and IL-6M forms before the functional hexamer. Due to its increased affinity for the IL-6R but its decreased ability to couple with gp130, we suggest that a stable IL-6 dimer may be an efficient IL-6 antagonist.

Antigens, CD↗

PubMed1996

Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor.

Amino Acid Sequence↗

PubMed1996

The interleukin-6 (IL-6) partial antagonist (Q159E,T162P)IL-6 interacts with the IL-6 receptor and gp130 but fails to induce a stable hexameric receptor complex.

The extracellular "soluble" domains of the IL-6 receptor (sIL-6R) and gp130 (sgp130) form a hexameric ternary receptor complex together with IL-6, consisting of two molecules of each component. In this report we have investigated the interactions of the partial IL-6 antagonist (Q159E,T162P)IL-6 ((QT)IL-6), with the sIL-6R and sgp130. The kinetic rate constants of the binding of sIL-6R to immobilized monomeric (QT)IL-6 or IL-6 were obtained using an optical biosensor with analysis of the primary data by linear and nonlinear regression. Both methods of analysis showed that, due to a higher off-rate, sIL-6R has lower apparent affinity for (QT)IL-6 than IL-6. The lower affinity of (QT)IL-6 was further confirmed by equilibrium binding measurements at the sensor surface and in solution. Using the biosensor it was also shown that the (QT)IL-6 complex interacts with sgp130, supporting the notion that the biological activity of (QT)IL-6 is mediated via gp130. However, the IL-6 mutant, when incubated with sIL-6R and sgp130, failed to induce a stable hexameric receptor complex, as shown by narrowbore size exclusion chromatography.

Antibodies, Monoclonal↗

PubMed1995

A mutation in the epidermal growth factor receptor in waved-2 mice has a profound effect on receptor biochemistry that results in impaired lactation.

The mutant mouse waved-2 (wa-2) is strikingly similar to transforming growth factor alpha-deficient mice generated by gene targeting in embryonic stem cells. We confirm that wa-2 is a point mutation (T-->G resulting in a valine-->glycine substitution at residue 743) in the gene encoding the epidermal growth factor (EGF) receptor. wa-2 fibroblastic cells lack high-affinity binding sites for EGF, and the rate of internalization of EGF is retarded. Although the tyrosine kinase activity of wa-2 EGF receptors is significantly impaired, NIH 3T3 cells lacking endogenous EGF receptors but overexpressing recombinant wa-2 EGF receptor cDNA are mitogenically responsive to EGF. While young and adult wa-2 mice are healthy and fertile, 35% of wa-2 mice born of homozygous wa-2 mothers die of malnutrition because of impaired maternal lactation.

3T3 Cells↗

PubMed1994

Affinity ranking of influenza neuraminidase mutants with monoclonal antibodies using an optical biosensor. Comparison with ELISA and slot blot assays.

A recently developed alternative to the more traditional techniques for studying antigen-antibody interactions has been examined. This method involves the use of an optical biosensor employing surface plasmon resonance detection. In this system one of the reactants is immobilized on the sensor surface and other reactants are passed over the sensor surface sequentially at a constant flow rate. Binding phenomena are detected in real time from changes in the angle at which surface plasmon resonance occurs. This is dependent, among other things, on changes in the refractive index (which is directly proportional to the mass) at or near to the sensor surface. Applications of this biosensor technique for comparing the binding of related neuraminidases, purified from escape mutants of influenza virus NWS/G70C/75 (N9), to two antibody Fab fragments, are described. These results were compared with those obtained from ELISA and slot blot assays on the same neuraminidases interacting with the same two monoclonal antibodies. The biosensor method was shown to be highly specific, permitting rapid screening of binding in such antigen-antibody systems.

Antibodies, Monoclonal↗

PubMed1994

Structure-function relationships for the EGF/TGF-alpha family of mitogens.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) are ligands for the EGF-receptor and act as mitogens for a variety of tissues. TGF-alpha, in particular, has been implicated as an autocrine growth factor for several cancer cell lines. Over the last 10 years many groups have examined the structure-function relationships in EGF/TGF-alpha in attempts to develop antagonists or agonists. In this review the results of these studies are summarised and related to the three-dimensional structure of EGF/TGF-alpha. The difficulties associated with the purification and characterisation of analogues of EGF/TGF-alpha and with the biological assays are discussed. It is clear that these difficulties have, in some cases, led to apparently contradicting results. The available binding data indicate that the receptor interaction surface for EGF/TGF-alpha might encompass one complete side of the molecule with a few strong binding determinants, in particular Arg41 and Leu47. The arginine at position 41 is the most critical residue and its full hydrogen-bonding capacity is needed for strong binding of EGF/TGF-alpha to the EGF-receptor. As this side of the molecule consists of residues from both the N- and C-terminal domain, it seems unlikely that agonists or antagonists can be developed on the basis of short peptides taken from the primary sequence. This concept is supported by the available binding and activity data.

Amino Acid Sequence↗

PubMed1993

Verification of the interaction between peptide T and CD4 using surface plasmon resonance.

Peptide T is currently in phase II clinical trials for the treatment of AIDS-associated dementia. Its putative mode of action is inhibition of binding of the HIV envelope protein (gp120) to its cellular receptor (CD4), thus preventing viral infectivity and gp120-induced neuronal toxicity. However, a number of reports have appeared in the literature which have failed to observe any inhibitory activity of Peptide T on CD4-gp120 binding, thus casting doubt on this hypothesis. This study uses a novel biosensor technique to demonstrate that Peptide T does bind to CD4 and that this binding can be specifically inhibited by an anti-CD4 monoclonal antibody. A detailed analysis of the kinetics of the interaction is presented.

Amino Acid Sequence↗

PubMed1993

Structural characterisation of native and recombinant forms of the neurotrophic cytokine MK.

The retinoic acid (RA)-inducible midkine (MK) gene encodes a heparin-binding protein which can induce neurite outgrowth in cultured mammalian embryonic brain cells. This cytokine shares 65% amino acid sequence identity with another RA-inducible cytokine, pleiotropin (PTN). Both proteins contain 10 conserved cysteine residues, all of which appear to be disulphide linked. MK and PTN are also rich in lysine and arginine residues rendering them susceptible to proteolysis during purification, and making large-scale preparation of these molecules inherently difficult. Recombinant MK has been expressed as a fusion protein using a pGEX vector transfected into E. coli. To enable refolding of MK, the fusion protein was stored in solution at 4 degrees C for 14 days in the presence of dithiothreitol (DTT). Thrombin cleavage of the fusion protein, post storage, typically generated 5 mg of MK per litre of bacterial pellet. To establish the structural integrity of the recombinant product, we have analysed the refolding kinetics and compared the disulphide bond assignment of recombinant MK with that of native MK and native PTN. The synergistic use of micropreparative HPLC, to separate and recover in small eluant volumes enzymatically derived peptide fragments, with matrix assisted laser desorption mass spectrometry (MALD-MS) and N-terminal sequence analysis has allowed the unambiguous identification of the disulphide bonded fragments of native and recombinant MK. The disulphide bond assignment of MK is C12-C36, C20-C45, C27-C49, C59-C91 and C69-C101, and is equivalent to that of PTN.

Amino Acid Sequence↗

PubMed1993

Mice with a null mutation of the TGF alpha gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation.

Mice homozygous for a disrupted transforming growth factor alpha (TGF alpha) gene are healthy and fertile, although some older mice show evidence of corneal inflammation. In contrast with TGF alpha +/- and +/+ animals, TGF alpha -/- mice have a pronounced waviness of the coat. Histological examination of the skin from TGF alpha -/- mice reveals a dramatic derangement of hair follicles. Mice with a disrupted TGF alpha gene also have curly whiskers, first evident on the day of birth. The phenotype of TGF alpha -/- mice is remarkably similar to that of the mouse mutant waved-1 (wa-1). Offspring resulting from crosses between TGF alpha -/- and wa-1 mice display the curly whisker-coat phenotype, indicating that the basis of the wa-1 phenotype is a mutation in the TGF alpha gene. These observations suggest that TGF alpha plays a pivotal role in determining skin architecture and in regulating hair development.

Animals↗

PubMed1992

The purification of a Rap1 GTPase-activating protein from bovine brain cytosol.

Two GTPase-activating proteins (GAPs) have been detected in extracts from bovine brain: GAP-1, which is specific for the activation of ras GTPases, and GAP-3, which is specific for the activation of the rap1 GTPases. We present a strategy for the purification to homogeneity of a cytosolic form of GAP-3 from bovine brain. The 100,000 x g supernatant from homogenized brains was chromatographed sequentially on DEAE Fast Flow, green H-E4BD Sepharose, Bio-Gel A1.5, hydroxyapatite, and phenyl-Sepharose prior to high resolution separation on Mono Q HR 5/5, phenyl-Superose HR 5/5, Mono Q PC 1.6/5, and Superose 12 PC 3.2/30. This procedure resulted in an approximately 18,000-fold purification, yielding 50 micrograms of GAP-3 from 1.6 kg of tissue. Purified cytosolic GAP-3 migrated as a single band of apparent Mr 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, on gel filtration cytosolic GAP-3 chromatographed as a dimer with an apparent Mr 92,000. Purified GAP-3 does not activate ras or rho GTPases and possesses no intrinsic GTPase activity. Amino acid sequence data indicated a proline-rich N terminus. The amino acid sequences of peptides generated by Staphylococcus aureus V8 digestion of reduced and pyridine-ethylated GAP-3 showed no similarity to the predicted primary structure of GAP-1 or any other proteins in the nucleic acid or protein data bases. By comparison with the data of Rubinfeld et al. (Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L., Watt, K., Crosier, W.J., McCormick, F., and Polakis, P. (1991) Cell 65, 1033-1042), it appears that the membrane-associated (Mr 85,000-95,000) and cytosolic forms of GAP-3 are derived from equivalent, or closely related, genes.

Amino Acid Sequence↗