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PubMed2005

Characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow.

OBJECTIVE: Adult bone marrow (BM) mesenchymal stem/progenitor cells (MS/PC) are a potentially useful tool for cell therapy and tissue repair. However, the identification of cell subsets rich in MS/PC from fresh BM has not been described. We have developed a means of identifying such subsets from untouched bone marrow. MATERIAL AND METHODS: First, MS/PC were enriched by short-time adherence (D(1-3)) before any cell division to evaluate the efficiency of CD73, CD105, CDw90, and CD49a antigens to select highly purified CD45(-)CD14(-) fluorescence-activated sorted subsets enriched in clonogenic mesenchymal cells. Then, we adapted this method to unmanipulated BM mononuclear cells (MNC). RESULTS: Short-time (D(1-3)) adherent CD45(-)CD14(-) cells expressing CD73 or CD49a antigens contained all the CFU-F, even though the CD105(+) and CDw90(+) subsets comprised less than half the total. In fresh unmanipulated BM MNC, CD73 and CD49a were also highly discriminative and allowed up to a 3 log enrichment of CFU-F when compared to BM MNC. Normal culture conditions upregulated most of the tested antigens. CONCLUSION: The CD45(-)CD14(-)/CD73(+) and CD45(-)CD14(-)/CD49a(+) phenotypes identified subsets containing all the CFU-F and sufficiently enriched to detect them in fresh BM, enabling evaluation of mesenchymal content of BM collections for cell therapy.

Adult↗

PubMed2004

Regulating eukaryotic gene expression with aptamers.

Aptamers are RNA or DNA oligonucleotides identified within a randomly synthesized library, through an in vitro selection procedure. The selected candidates display a pre-determined property of interest with respect to a given target. Successful selection has been carried out against targets ranging from small (amino acids, antibiotics) to macro-molecules (proteins, nucleic acids). They generally show an affinity in the nanomolar range and a high specificity of target recognition. Interestingly, aptamers selected against purified targets in the test tube retain their properties within cells. RNA aptamers can be generated in situ from an appropriate DNA construct or delivered as nuclease-resistant oligonucleotide analogues. For example, aptamers recognizing RNA structure through loop-loop interactions modulate the trans-activation of in vitro transcription mediated by the TAR RNA element of human immunodeficiency virus type 1. Consequently, they constitute both exquisite tools for functional genomics analysis and promising prototypes of therapeutic agents. Natural aptameric motifs have been identified within mRNA sequences, which upon binding to a metabolite control the expression of the encoded gene, which is generally involved in the biosynthesis of this particular metabolite.

Amino Acid Motifs↗

PubMed2004

Characterization of the Anaplasma marginale msp2 locus and its synteny with the omp1/p30 loci of Ehrlichia chaffeensis and E. canis.

Major surface protein 2 (MSP2) is an immunodominant and antigenically variant protein in the outer membrane of the rickettsia Anaplasma marginale. MSP2 variation is generated by recombination into a single operon-linked genomic expression site. The complete 5.6-kb msp2 locus was identified by sequencing a 90-kb region of the St. Maries strain of A. marginale. The locus encoded, in a 5' to 3' direction, a transcriptional regulator followed by five outer membrane proteins, OMP1, OpAG3, OpAG2, OpAG1, and MSP2. The sequences of this entire locus were analyzed using six genetically and phenotypically distinct strains of A. marginale. The overall locus structure was highly conserved with 100% identity among strains in the transcriptional regulator. Synonymous and nonsynonymous exchanges were infrequent in omp1 and rare in opag1 and opag2 among the six strains without strong bias for either type of exchange (neutral mutations). In contrast, mutations in opag3 seem to underlie purifying (negative) selection reflecting pressure to retain protein structure, in marked contrast to the highly antigenically variant MSP2. Interestingly, the 5' structure of this A. marginale msp2 locus is conserved in the omp1 gene locus of Ehrlichia chaffeensis and p30 gene locus of E. canis despite marked divergence between genera in the structure of the 3' region of the loci. This supports the hypothesis that the expression sites of these important immunogenic proteins are derived from a common precursor with later divergent evolution along genus lines.

Anaplasma marginale↗

PubMed2000

Binding of detergents and inhibitors to bovine complex I - a novel purification procedure for bovine complex I retaining full inhibitor sensitivity.

Mitochondrial complex I exhibits some peculiar and poorly understood features regarding the effects of detergents on activity and sensitivity to hydrophobic inhibitors that are not seen with other membrane complexes using ubiquinone as a substrate. Therefore, we investigated the interaction of complex I from bovine heart mitochondria with different types of detergents by monitoring activity, degree of inhibition and inhibitor binding in the presence of increasing concentrations of detergent. It is shown that apart from their nature as solubilizing and delipidating agents the polyoxyethylene-ether detergents Triton X-100, Brij-35 and Thesit act as specific inhibitors of complex I and compete with classical complex I inhibitors for a common binding domain. These findings were used to develop a novel large-scale chromatographic procedure for isolation of inhibitor-sensitive NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxylapatite, ion-exchange and gel-exclusion chromatography. By switching detergents from Triton X-100 to dodecylmaltoside after hydroxylapatite chromatography the procedure yields highly pure, monodisperse and fully inhibitor-sensitive enzyme.

Amines↗

PubMed1999

Polyethylene glycol-mediated infection of non-permissive mammalian cells with semliki forest virus: application to signal transduction studies.

Semliki Forest Virus (SFV) vectors allow the subcloning of a gene of interest directly in the expression vector, thus avoiding the need to select and purify viral recombinants, making this viral expression system attractive over many others for mammalian protein expression. We now describe a novel and generally applicable method for infection of non-permissive mammalian cells with SFV, that greatly enhances the utility of this expression system. We demonstrate that the hygroscopic polymer poly (ethylene glycol), PEG, promotes the infectivity of cells by SFV under conditions that did not promote cell-cell fusion. We also found that the PEG-induced infection and expression of an exogenous protein (green fluorescent protein, GFP) did not elevate the basal tyrosine kinase activity, induce a stress-activated responses, or result in aberrant cell responses. Expression of GFP tagged-Vav, an activator of stress-activated protein kinase (SAPK/JNK), resulted in the expected induction of JNK activity and in the normal redistribution of Vav in response to engagement of the high affinity receptor for IgE (FcepsilonRI). Thus, our findings that PEG allows the infection of non-permissive cells by SFV makes this system extremely attractive for expression of proteins in mammalian cells, and studies on signal transduction and cellular localization in immune and non-immune cells.

3T3 Cells↗

PubMedDate not supplied

Some structural studies on the galactose-containing polysaccharide from bovine placenta.

Polysaccharides were extracted from 8-month-old placenta with aqueous HgCl2. The protein-free material was purified by selective precipitation with Cetavlon in the presence of sodium borate at pH 8.5 and was homogeneous on molecular-sieve chromatography, electrophoresis, and on treatment with Concanavalin A. The preparation contained galactose and glucose as principal monosaccharides with 5 per cent of hexosamines. Methylation studies suggested that D-gluco and D-galactopyranosyl units may be constituents of glucan and galactan respectively which form a molecular aggregate that does not dissociate during the fractionation procedures. After treatment of the fraction with beta-amylase, the proportion of glucose in the polysaccharide diminished, indicating the presence of (1-->4)-linked alpha-D-glucopyranosyl residues. Also, when the fraction was treated with a crude protease having glucosidase activity a residual alpha-D-galactopyranan was isolated and found to contain non-reducing end-groups (30.0 per cent), 3-O-(39.5 per cent) and 3,6-di-O-substituted (30.5 per cent) units. The structure of the galactan was not modified according to methylation data, on removal of the glucosyl component. The polysaccharide fraction (pH 8.5 Cetavlon), isolated from bovine placenta, thus contains a glycogen-like material associated with a galactan as molecular aggregate. This galactan has not been previously recognized in bovine placenta and its occurrence in this organ supports the hypothesis that galactose-containing polysaccharides are involved in foetal development.

Animals↗

PubMed2003

Time course of T-cell responses to MOG and MBP in patients with clinically isolated syndromes.

CD4(+) T-cell lines (TCLs) from patients with clinically isolated syndromes (CIS) were selected with purified human myelin basic protein (MBP) and recombinant human myelin oligodendrocyte glycoprotein (rhMOG), at onset of neurological symptoms and when patients developed clinically definite multiple sclerosis (CDMS). The epitope specificity of each TCL was mapped with overlapping synthetic peptides. TCLs were assessed for their ability to secrete IFN-gamma, IL-4, and IL-6. Diverse patterns of epitope recognition were observed: (a) recognition of a broad spectrum of MBP peptide epitopes with evidence of shifts over time; (b) an initial T-cell response focused to a restricted segment of the MBP molecule (83-102) that broadened over the course of disease; and (c) persistence of a focused anti-MOG T-cell response. CIS patients who failed to develop CDMS maintained a focused epitope response against two to six MBP epitopes. Most MBP peptide-specific TCLs secreted considerable amounts of IFN-gamma and low amounts of IL-4 and IL-6, whereas anti rhMOG(Igd) peptide-specific TCLs secreted preferentially IL-4 and IL-6. These data raise important issues for the pathogenesis and treatment of multiple sclerosis (MS).

Adolescent↗

PubMed2002

Differential expression and genetic variation of hepatic messenger RNAs from genetically lean and fat chickens.

Although excessive adiposity has become a major drawback in meat type chicken production, few of the genes involved in this process have been characterized so far. In order to identify putative genes involved in adiposity, we performed differential display analysis of RNAs extracted from the liver of divergently selected lean and fat chickens. Twenty-six differential products were selected and purified by single strand conformation polymorphism gel electrophoresis before sequencing and Northern blot analyses. An orthologous sequence of a mammalian cytochrome P450 2C subfamily member was proven to be differentially expressed in the liver of lean and fat chickens and could play an important role in the regulation of adiposity. In mammals, these genes are involved in detoxification of xenobiotics and metabolism of some important biological compounds. Four other genes were found differentially expressed to a lower extent. Some unidentified products were shown to be lean or fat specific, with sequence polymorphism and liver specific expression, strongly suggesting that the related gene could be directly involved in adiposity. Our data indicate that differential display can evidence genes with differential expression and with sequence polymorphism, making this strategy more accurate for differential analysis of messenger RNAs.

Adipose Tissue↗

PubMed1986

Membrane antigen expression during hemopoietic differentiation.

The pattern of certain groups of antigens expressed on the surface of hemopoietic cells changes either during the course of differentiation from pluripotent stem cells to mature functional cells or as a function of the proliferative state of the cells. A map of these changes is emerging and is providing valuable information for selecting and purifying rare stem cells and for classifying the acute leukemias. This knowledge is also beginning to provide insights into physiological and pathological cellular interactions affecting the early stages of hemopoiesis, and is being exploited to remove T lymphocytes from allogeneic bone marrow grafts in order to prevent graft-vs.-host disease as well as leukemic cells from bone marrow before autologous reinfusion. In this article I will briefly review the cellular basis of hemopoiesis and then discuss the methods used to determine the presence of antigens on normal hemopoietic cells. I will then summarize the pattern of membrane antigens expressed during differentiation and conclude by discussing the biological and therapeutic implications.

Animals↗

PubMed2000

New approaches for cell-specific targeting: identification of cell-selective peptides from combinatorial libraries.

Peptides that recognize specific cell types promise to be valuable tools both in research and clinical applications. Cell-specific peptides can be useful as drug delivery vehicles, diagnostic agents, affinity reagents for cell purification, gene therapy delivery agents, and research tools to probe the nature of a cell's surface. Recently, cell-specific targeting-peptides have been identified by phage-display selections against purified cell-surface markers, whole cells in tissue culture, and even tissues within live animals. These methods for identifying cell-targeting peptides will certainly increase the tools available to the scientist for cell-specific targeting.

Animals↗

PubMed1996

Iterative optimization of high-affinity protease inhibitors using phage display. 2. Plasma kallikrein and thrombin.

As discussed in the accompanying paper [Markland, W., Ley, A. C., & Ladner, R. C. (1996) Biochemistry 35, 8045-8057], we generated libraries from the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1) using multivalent M13 III display and derived potent inhibitors of human plasmin (PLA) by iterative variegation and selection. Here, we show that high-affinity, high-specificity binders to human plasma kallikrein (pKAL) and human thrombin (THBN) can be obtained starting from the identical library and employing the same iterative variegation procedures used to obtain PLA inhibitors. Lib#1 (allowing 31 200 variants involving five positions near the P1 residue of LACI-D1) and its pKAL-biased derivative, Lib#4 (allowing an additional 1600 variants at residues 31, 32, 34, and 39), were screened against pKAL, yielding potent inhibitors. One of these, EPI-K401, has Ki = 284 pM, very high specificity, and excellent stability. We used information from Lib#4 selectants to design Lib#5 (allowing 1.5 x 10(6) amino-acid sequences involving nine varied positions) from which we obtained an inhibitor (EPI-K503) having high affinity for pKAL (Ki = 40 pM) and retaining the high specificity of EPI-K401. When we screened Lib#1 and its THBN-tailored derivative, Lib#6, against THBN, we obtained a different and very homogeneous population of selected molecules. The purified proteins derived from Lib#6 selectants bound to THBN-agarose beads but did not inhibit proteolytic activity of THBN, suggesting that these selectants bind to a site on THBN other than the catalytic site. Thus, a single large combinatorial library can serve as a source to obtain highly specific, high-affinity binding molecules for each of several targets. Furthermore, the results with THBN show that the binding of Kunitz domains to other proteins is not limited to the catalytic sites of trypsin-homologous proteases.

Amino Acid Sequence↗

PubMed2001

Polyoxometalate HIV-1 protease inhibitors. A new mode of protease inhibition.

Nb-containing polyoxometalates (POMs) of the Wells-Dawson class inhibit HIV-1 protease (HIV-1P) by a new mode based on kinetics, binding, and molecular modeling studies. Reaction of alpha(1)-K(9)Li[P(2)W(17)O(61)] or alpha(2)-K(10)[P(2)W(17)O(61)] with aqueous H(2)O(2) solutions of K(7)H[Nb(6)O(19)] followed by treatment with HCl and KCl and then crystallization affords the complexes alpha(1)-K(7)[P(2)W(17)(NbO(2))O(61)] (alpha(1)()1) and alpha(2)-K(7)[P(2)W(17)(NbO(2))O(61)] (alpha(2)()1) in 63 and 86% isolated yields, respectively. Thermolysis of the crude peroxoniobium compounds (72-96 h in refluxing H(2)O) prior to treatment with KCl converts the peroxoniobium compounds to the corresponding polyoxometalates (POMs), alpha(1)-K(7)[P(2)W(17)NbO(62)] (alpha(1)()2) and alpha(2)-K(7)[P(2)W(17)NbO(62)] (alpha(2)()2), in moderate yields (66 and 52%, respectively). The identity and high purity of all four compounds were confirmed by (31)P NMR and (183)W NMR. The acid-induced dimerization of the oxo complexes differentiates sterically between the cap (alpha(2)) site and the belt (alpha(1)) site in the Wells-Dawson structure (alpha(2)()2 dimerizes in high yield; alpha(1)()2 does not). All four POMs exhibit high activity in cell culture against HIV-1 (EC(50) values of 0.17-0.83 microM), are minimally toxic (IC(50) values of 50 to >100 microM), and selectively inhibit purified HIV-1 protease (HIV-1P) (IC(50) values for alpha(1)()1, alpha(2)()1, alpha(1)()2, and alpha(2)()2 of 2.0, 1.2, 1.5, and 1.8 microM, respectively). Thus, theoretical, binding, and kinetics studies of the POM/HIV-1P interaction(s) were conducted. Parameters for [P(2)W(17)NbO(62)](7)(-) were determined for the Kollman all-atom (KAA) force field in Sybyl 6.2. Charges for the POM were obtained from natural population analysis (NPA) at the HF/LANL2DZ level of theory. AutoDock 2.2 was used to explore possible binding locations for the POM with HIV-1P. These computational studies strongly suggest that the POMs function not by binding to the active site of HIV-1P, the mode of inhibition of all other HIV-1P protease inhibitors, but by binding to a cationic pocket on the "hinge" region of the flaps covering the active site (2 POMs and cationic pockets per active homodimer of HIV-1P). The kinetics and binding studies, conducted after the molecular modeling, are both in remarkable agreement with the modeling results: 2 POMs bind per HIV-1P homodimer with high affinities (K(i) = 1.1 +/- 0.5 and 4.1 +/- 1.8 nM in 0.1 and 1.0 M NaCl, respectively) and inhibition is noncompetitive (k(cat) but not K(m) is affected by the POM concentration).

Anti-HIV Agents↗

PubMed2001

A calcium-binding protein with similarity to serum albumin localized to the ER-Golgi network and cell walls of spinach (Spinacia oleracea).

Using polyclonal antibodies raised against human serum albumin (HSA), a 70-kDa microsomal protein with an isoelectric point of approximately 6.5 was detected in spinach (Spinacia oleracea L.). The protein was purified by selective ammonium sulfate precipitation and anion exchange HPLC. The protein shared 100% identity with the first 15 amino acids at the NH2 terminus of HSA, including the X-X-H amino acid region, which was identified in HSA as being responsible for binding of copper, zinc, indole derivatives and calcium. Blue staining of the protein with the cationic carbocyanine dye 'Stains-all' and 45Ca overlay following SDS-PAGE also suggest that the 70-kDa plant protein binds calcium. The protein reacted positively with carbohydrate specific thymol stain, and the carbohydrates associated with the protein were identified by gas chromatography-mass spectrometry (GC-MS) as galactose and galacturonic acid. The 70-kDa plant protein was present in the detergent-poor phase following Triton X-114 extraction of the microsomal proteins. Cell fractionation using continuous sucrose gradients showed that the protein is present in membrane fractions with high activity of endoplasmic reticulum (ER) and Golgi marker enzymes. Using nitrocellulose tissue prints probed with anti-HSA antibodies, we demonstrated that the protein is present in the apoplastic space of petioles, suggesting that the protein is secreted to the apoplast of cortex cells in plants. Localization and binding properties suggest that the plant protein identified in the present study may participate in secretion processes, possibly involved with the transport of precursors required for cell-wall synthesis.

Journal Article↗

PubMed2000

Calcium-sensing receptor expression and signalling in human parathyroid adenomas and primary hyperplasia.

OBJECTIVE: Both in vivo and in vitro evidence indicates that primary hyperparathyroidism is characterized by a reduced sensitivity to extracellular calcium ([Ca2+]o). The existence of alterations in the expression and signalling of calcium sensing receptor (CaSR) in parathyroid neoplasia is still uncertain. In order to clarify the role of CaSR in the reduced [Ca2+]o sensing of parathyroid neoplasia we investigated PTH secretion and intracellular effectors triggered by CaSR activation as well as the levels of expression of CaSR and CaSR coupled G proteins (Gq/G11) in parathyroid adenomas and primary hyperplasia. MATERIALS AND METHODS: The study included 27 parathyroid adenomas, 4 cases of primary hyperplasia and pools of normal parathyroid biopsies. Tissues were either snap frozen in liquid nitrogen or placed in sterile medium for cell dispersion. The effects of increasing [Ca2+]o on in vitro PTH release, intracellular cAMP levels and intracellular calcium ([Ca2+]i) in cells loaded with the Ca2 + indicator fura-2 were evaluated. CaSR mRNA levels were assessed by semiquantitative RT-PCR analysis, using GAPDH as internal standard, while CaSR protein was detected by western blot analysis using a specific polyclonal antibody. Purified antisera selective for G11alpha and Gqalpha were used to detect this class of proteins. RESULTS: In basal conditions (at 0.5 mM [Ca2+]o) in vitro PTH released ranged from 29.4 to 1186 pg/well/60 minutes. Increasing [Ca2+]o from 0.5 to 1, 2.5 and 5 mM caused a variable effect. One group (n = 7) showed a significant but partial reduction of PTH release (of 17 to 60% of basal levels) that occurred at physiological [Ca2+]o concentrations (1 mM) while the remainder showed either inhibition detectable only at 2.5 mM (n = 15) or total (n = 9) resistance to [Ca2+]o. In the responsive cells, [Ca2+]o (1-5 mM) caused a pertussis toxin-insensitive [Ca2+]i rise (ranging from 10% to 260%), due to Ca2+ release from intracellular stores, and an inhibition of forskolin-stimulated cAMP levels. By RT-PCR almost all tumours tested showed a substantial reduction in CaSR mRNA levels when compared to the normal tissue (CaSR/GAPDH ratio: 3.1 +/- 0.5 vs. 15.5 +/- 3.1; P < 0.001), which was confirmed by immunoblotting analysis demonstrating low levels of CaSR protein in tumour tissues. Moreover, low amounts of G11alpha and Gqalpha, the G proteins involved in CaSR coupling, were observed in the majority of pathological tissues. CONCLUSIONS: The study shows that the activation of the calcium sensing receptors expressed in adenomatous parathyroid glands modulates intracellular effectors in a similar way to those operating in the normal parathyroid. Although a reduction of calcium sensing receptor expression is probably involved in the poor inhibition of PTH release induced by [Ca2+]o, this is not the only factor altering [Ca2+]o sensing in parathyroid adenomas, since tumours characterized by different in vitro sensitivity to [Ca2+]o showed similar CaSR levels. The low content of G proteins of the Gq subfamily might represent an additional alteration leading to a defective [Ca2+]o sensing.

Adenoma↗

PubMed1974

Cell-free translation of messenger RNA of simian virus 40: synthesis of the major capsid protein.

Extracts of wheat germ are capable of synthesizing the major capsid protein of simian virus 40. Poly(A)-containing RNA from BS-C-1 cells infected with simian virus 40 directed the synthesis of a novel polypeptide that migrates in polyacrylamide gels together with the major capsid polypeptide of simian virus 40, VP-1. The patterns of the major tryptic peptides of purified VP-1 and the novel polypeptide synthesized in vitro were identical after two-dimensional paper electrophoresis. The novel polypeptide was not synthesized in response to poly(A)-rich RNA from uninfected cells or from virus-infected cells treated with cytosine arabinoside. Messenger RNA from infected cells purified by selective hybridization to DNA of simian virus 40 directs the synthesis of a major polypeptide of electrophoretic mobility similar to that of VP-1 of simian virus 40. This approach should prove useful in identifying additional products specified by DNA tumor viruses.

Adenine Nucleotides↗

PubMed1979

Inhibition of human neuroblastoma DNA polymerase activities by plant lectins and toxins.

The effects of concanavalin A and ricin (RCAII, Mr 65,000) on [3H]thymidine incorporation into human neuroblastoma IMR-32 DNA showed reduction of total DNA synthesis to 50% and 70% of control, respectively. Two DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7.) activities (alpha and beta) involved in the biosynthesis in vitro of DNA were separated by sucrose density gradient centrifugation from IMR-32 cell homogenate. The DNA polymerase alpha activity was also purified by selective precipitation with polyethylene glycol (Mr 6000) followed by agarose-concanavalin A column chromatography. The activities of both DNA polymerases were examined at various concentrations of mutagenic and nonmutagenic plant agglutinins and the toxin ricin. Concanavalin A and ricin specifically inhibited DNA polymerase alpha activity (activity reduced to 19% and 10%, respectively), whereas DNA polymerase beta activity was inhibited (reduced to 16%) by red kidney bean agglutinin (PHA-P).

Cell Line↗

PubMed1986

A low-molecular-weight RNA from mouse ascites cells that hybridizes to both 18S rRNA and mRNA sequences.

A low-molecular-weight RNA species from mouse ascites cells has been selected and purified by its intermolecular RNA X RNA hybridization capabilities. This 4.5S RNA is able to base pair with poly(A)+ mRNA sequences and with 18S rRNA. Melting experiments have shown that the intermolecular hybrids formed with this complementary low-molecular-weight RNA are of comparable stability to other RNA X RNA interactions. Analysis has shown that this hybridizing RNA is 87 nucleotides long and has an unusual sequence structure. Located near the 3' terminus is an alternating pyrimidine dinucleotide region of UUCCUUCCUU. This region along with the 3'-adjacent nucleotides form a 14-nucleotide sequence that exhibits perfect complementarity with 18S rRNA. An additional region of 10 nucleotides at the 3' terminus is perfectly homologous to a similarly located sequence in 5.8S rRNA. An obvious RNA polymerase III binding site is not found internally in this low-molecular-weight RNA sequence. The complementary and homologous character of hybridizing RNA with respect to rRNA and mRNA sequences suggests a potential regulatory role for this RNA in the coupling of ribosome and mRNA functions.

Animals↗

PubMed1990

Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma.

We have investigated human gliomas that amplify and rearrange the epidermal growth factor receptor gene, with generation of an in-frame deletion mutation of 802 nucleotides in the external domain. This in-frame deletion mutation generates a local amino acid sequence at the fusion junction of what normally were distant polypeptide sequences in the intact epidermal growth factor receptor. This 14-amino acid peptide was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion junction peptide antibody was purified by passage of the antiserum over a peptide affinity column with acidic elution. The purified antibody selectively bound the glioma deletion mutant as compared to the intact epidermal growth factor receptor as assessed by immunocytochemistry, immunofluorescence, immunoprecipitation with gel electrophoresis, and binding experiments using radioiodinated antibody. These data indicate that it is feasible to generate site-specific anti-peptide antibodies that are highly selective for mutant proteins in human tumors. The anti-peptide antibody described here, and other mutation site-specific antibodies, should be ideal candidates for tumor immunoimaging and immunotherapy.

Amino Acid Sequence↗