Biomedical subjects

I Clark-Lewis

Publications and source records attributed to I Clark-Lewis.

At least 127 records · Page 7Linked to original sources

PubMed1991

Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis.

Several structural homologues of the chemotactic peptide neutrophil-activating peptide 1/IL-8 (NAP-1/IL-8) were tested for their ability to influence the expression and function of adhesion-promoting receptors on human polymorphonuclear leukocytes (PMN). NAP-2, melanoma growth stimulatory activity, and two forms of NAP-1/IL-8 (ser-NAP-1/IL-8 and ala-NAP-1/IL-8, consisting of 72 and 77 amino acids, respectively), each caused an increase in the expression of CD11b/CD18 (CR3) and CR1, which was accompanied by a decrease in the expression of leukocyte adhesion molecule-1 (LAM-1, LECAM-1). The binding activity of CD11b/CD18 was also enhanced 3- to 10-fold by these peptides, but enhanced function was transient: binding of erythrocytes coated with C3bi reached a maximum by 30 min and declined thereafter. Ser-NAP-1/IL-8, ala-NAP-1/IL-8, NAP-2, and melanoma growth stimulatory activity also caused a two- to threefold enhancement of the phagocytosis of IgG-coated erythrocytes (EIgG) by PMN without causing a large increase in the expression of Fc gamma receptors. Enhanced phagocytosis of EIgG appeared to be mediated through CD11b/CD18, because F(ab')2 fragments of an antibody directed against CD18 inhibited NAP-1/IL-8-stimulated ingestion of EIgG. The four active peptides caused a rapid, transient increase in the amount of F-actin within PMN, indicating that they are capable of influencing the structure of the microfilamentous cytoskeleton, which participates in phagocytosis. Two other NAP-1/IL-8-related peptides, platelet factor 4 and connective tissue-activating peptide III, were without effect on expression of CD11b/CD18, CR1, and LAM-1, binding activity of CD11b/CD18, or Fc-mediated phagocytosis, and increased actin polymerization only slightly. Our observations indicate that several members of the NAP-1/IL-8 family of peptides were capable of promoting integrin-mediated adhesion and Fc-mediated phagocytosis, processes important in the recruitment of PMN to sites of inflammation and antimicrobial responses of PMN.

Actins↗

PubMed1991

Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities.

Interleukin-8 (IL-8) is an inflammatory mediator that stimulates neutrophil migration and functional activation. Analogs of human IL-8 were chemically synthesized, purified, and compared with the full-length 72-residue synthetic IL-8 for their ability to stimulate neutrophil chemotaxis and exocytosis as measured by assaying for release of elastase, as well as their binding to specific receptors in competition assays. Analogs corresponding to the less abundant natural forms, 3-72, 4-72, and 77-residue IL-8, were evaluated and the 3-72 and 4-72 had 2-5-fold higher potencies, whereas the 77-residue IL-8 was 2-fold less potent. A major finding was that NH2-terminal residues 4, 5, and 6 were absolutely essential for IL-8 activity and receptor binding. Quantitative dissociation of elastase release and chemotaxis activity was detected with 5-72, which compared with 1-72, was 80-fold less potent in the elastase assay, but was only slightly less potent in stimulating chemotaxis. IL-8 6-72 lacked all the biological activities tested but had detectable receptor binding activity. The NH2-terminal peptide, AVLPRSAKEL, lacked activity and receptor binding, suggesting that the NH2-terminal region alone is not sufficient for function. Comparison of analogs shortened at the COOH terminus showed that potency was progressively reduced as the COOH-terminal residues were excluded. However activity was retained in an analog (1-51) with the entire COOH-terminal alpha helix and beta turn missing. A peptide corresponding to the COOH-terminal 22 residues, although inactive alone, synergized with the 1-51 analog in stimulating elastase release. The results suggest that the NH2-terminal residues 4, 5, and 6, which are disordered in the IL-8 solution structure, are directly involved in receptor binding, but the COOH-terminal alpha helix is probably important for stabilizing the three-dimensional structure. Other regions within residues 7-51 are also functionally important.

Amino Acid Sequence↗

PubMed1991

The cGMP-gated channel of the rod photoreceptor cell characterization and orientation of the amino terminus.

The molecular properties and orientation of the cGMP-gated cation channel of bovine rod outer segment membranes were studied using biochemical and immunochemical methods. Western blots labeled with anti-channel monoclonal antibodies indicate that the channel has a subunit Mr of 63,000 in bovine rod outer segment membranes prepared in the presence and absence of protease inhibitors and in rod outer segments from other mammalian retinas. The channel has an apparent Mr of 78,000 in both COS-1 cells and Xenopus oocytes expressing the cloned cDNA. NH2-terminal sequence analysis indicates that the lower Mr of the channel in rod outer segments is caused by the absence of the first 92 amino acids predicted by cDNA sequence analysis. Immunofluorescent and immunogold labeling has confirmed that the 63,000 form of the channel is present in rod outer segments. These results indicate that photoreceptor cell-specific co-translational or post-translational cleavage of the NH2-terminal segment of the channel occurs prior or during the incorporation of the channel into the rod outer segment plasma membrane. Immunogold labeling studies using site-directed antibodies also indicate that the NH2-terminal segment of the rod outer segment channel is exposed on the cytoplasmic side of the plasma membrane.

Amino Acid Sequence↗

PubMed1991

Platelet-activating factor- and thrombin-induced stimulation of p34cdc2-cyclin histone H1 kinase activity in platelets.

Numerous studies of cell cycle control in dividing cells have pointed to the central role of a 34-kDa histone H1 kinase (p34cdc2) complexed with regulatory subunits known as cyclins. We now report that p34cdc2-cyclin may also participate in signal transduction in nonproliferating, terminally differentiated cells, in this instance during sheep platelet activation. Immunological evidence for the presence of a p34cdc2 cognant in sheep platelet cytosol was obtained with antipeptide antibodies raised against peptide sequences in the conserved PSTAIRE and C-terminus regions of murine cdc2. The immunoreactive 32-kDa protein was adsorbed onto p13suc1-Sepharose, which selectively binds p34cdc2. A 58-kDa protein that also bound to p13suc1-Sepharose was identified as cyclin A on the basis of its size and immunoreactivity with two different anticyclin peptide antibodies. The p34cdc2-cyclin A complex was regulated during platelet activation. Its histone H1 phosphorylating activity was stimulated 2-fold in p13suc1-Sepharose extracts from platelets that had been exposed to platelet-activating factor or thrombin for 1 min prior to harvesting. Our findings imply that the p34cdc2-cyclin complex may serve alternative functions besides control of cell division.

Amino Acid Sequence↗

PubMed1991

Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase.

Synthetic peptides have been used to define the consensus amino acid sequence for substrate recognition by the meiosis-activated myelin basic protein (MBP) kinase (p44mpk), which was purified from maturing sea star oocytes. This protein kinase shares many properties with the mitogen-activated microtubule-associated protein-2 kinase (p42mapk) in vertebrates. Recently, Thr-97 in the tryptic fragment KNIVTPRTPPPSQGK of bovine MBP was identified as the major site of phosphorylation by p44mpk (Sanghera, J. S., Aebersold, R., Morrison, H. D., Bures, E. J., and Pelech, S. L. (1990) FEBS Lett. 273, 223-226). Synthetic peptides modeled after this sequence revealed that the presence of a proline residue C-terminal (+1 position) to the phosphorylatable threonine (or serine) residue was critical for recognition by p44mpk. Although not essential, a proline residue located at the -2 position enhanced the Vmax of peptide phosphorylation. Basic, acidic, and non-polar residues were equally tolerated at the -1 position. The presence of an amino acid residue at position -3 also increased peptide phosphorylation. Thus, the optimum consensus sequence for phosphorylation by p44mpk was defined as Pro-X-(Ser/Thr)-Pro, where X is a variable amino acid residue, but ideally not a Pro. Peptides that included this sequence were phosphorylated by p44mpk with Vmax values approaching 1 mumol.min-1.mg-1 and with apparent Km values of approximately 1 mM). Pseudosubstrate peptides in which the phosphorylatable residue was replaced by valine or alanine were weak inhibitors of p44mpk (apparent Ki values of approximately 3 mM). Over 40 distinct protein kinases contain Pro-X-(Ser/Thr)-Pro sequences including the human receptors for insulin and epidermal growth factor, and kinases encoded by the human proto-oncogenes abl, neu, and raf-1, and Schizosaccharomyces pombe cell cycle control genes ran-1 and wee-1. Multiple putative sites were also identified in rat microtubule-associated protein-2, human retinoblastoma protein, human tau protein, and Drosophila myb protein and RNA polymerase II.

Amino Acid Sequence↗

PubMed1991

Neutrophil-activating peptide 2 and gro/melanoma growth-stimulatory activity interact with neutrophil-activating peptide 1/interleukin 8 receptors on human neutrophils.

Neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8), neutrophil-activating peptide 2 (NAP-2), and gro/melanoma growth-stimulatory activity (gro/MGSA) are potent inflammatory cytokines with homologous structure and similar neutrophil-activating properties. Receptors on human neutrophils that interact with these peptides were studied. Analysis of 125I-NAP-1/IL-8 binding at 0-4 degrees C revealed 64,500 +/- 14,000 receptors/cell with an apparent Kd of 0.18 +/- 0.07 nM (mean +/- S.D. of six independent experiments). Unlabeled NAP-1/IL-8, NAP-2, and gro/MGSA competed with 125I-NAP-1/IL-8 for binding to human neutrophils. Competition with increasing concentrations of unlabeled NAP-2 and gro/MGSA resolved two classes of NAP-1/IL-8 binding sites: about 70% of them bound NAP-2 and gro/MGSA with high affinity (Kd: 0.34 +/- 0.2 and 0.14 +/- 0.02), while 30% were of low affinity (Kd: 100 +/- 20 and 130 +/- 10 nM). Different binding sites, however, were not apparent upon competition with unlabeled NAP-1/IL-8, suggesting that both classes of receptors have similar affinities for NAP-1/IL-8. The existence of two receptors was also suggested by ligand cross-linking and cross-desensitization experiments. Two neutrophil membrane proteins with apparent Mr of 66,000-74,000 and 42,000-46,000 became cross-linked to 125I-NAP-1/IL-8, and the labeling was decreased when excess NAP-1/IL-8, NAP-2, or gro/MGSA was present. Stimulation of neutrophils with NAP-1/IL-8 resulted in desensitization toward a subsequent challenge with NAP-2 or gro/MGSA as shown by the rise in cytosolic free calcium. By contrast, following primary stimulation with NAP-2 or gro/MGSA, responses to NAP-1/IL-8 were only moderately attenuated, supporting the existence of NAP-1/IL-8 receptors which bind NAP-2 or gro/MGSA with low affinity. In conclusion, our results demonstrate that NAP-2 and gro/MGSA act upon human neutrophils by directly interacting with two classes of receptors for NAP-1/IL-8.

Amino Acid Sequence↗

PubMed1991

Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide.

Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Studies of the small heat shock proteins of Caenorhabditis elegans using anti-peptide antibodies.

Peptides corresponding to selected regions of the 16 kDa small heat shock proteins (hsps) of the nematode C. elegans were synthesized and used to elicit polyclonal antibodies. It was found that these antibodies reacted predominantly with either the 16 kDa or the 18 kDa proteins, suggesting a close structural similarity between these hsps. Western blots of two-dimensional gels revealed extensive heterogeneity in these proteins, probably resulting from post-synthetic modifications. The native structures of both size classes of hsps were found to consist of large complexes of 4-5 x 10(5) Da.

Amino Acid Sequence↗

PubMed1991

[Ca2+]i changes and respiratory burst in human neutrophils and monocytes induced by NAP-1/interleukin-8, NAP-2, and gro/MGSA.

The stimulatory effects of neutrophil-activating peptide 1 (NAP-1), also termed interleukin 8 (IL-8), neutrophil-activating peptide 2 (NAP-2), and melanoma growth-stimulatory activity (gro/MGSA) on human neutrophils and monocytes were compared on the basis of two responses that can be assessed in real time, the changes in cytosolic free calcium and the respiratory burst. All three peptides induced a rapid and transient rise of cytosolic-free calcium and the respiratory burst in neutrophils. Both responses were also obtained in monocytes on stimulation with NAP-1/IL-8 and gro/MGSA, but not with NAP-2, which appeared to be more selective for neutrophils. Pretreatment with concanavalin A (ConA) enhanced several fold the rate and duration of the respiratory burst of neutrophils stimulated with all three peptides and of monocytes stimulated with NAP-1/IL-8 and gro/MGSA, but not with NAP-2. Sequential stimulation showed mutual cross desensitization by NAP-2 and gro/MGSA in neutrophils. In addition, desensitization of neutrophils toward NAP-2 and gro/MGSA, and of monocytes toward gro/MGSA, was obtained by prestimulation with NAP-1/IL-8. Prestimulation with either NAP-2 or gro/MGSA, however, did not desensitize the cells for NAP-1/IL-8. These results suggest that under conditions where multiple stimulatory agents are produced, neutrophil-activating peptides may contribute to the formation of substantial amounts of oxygen-derived radicals. In addition, the study shows that NAP-1/IL-8 and gro/MGSA, but not NAP-2, have some stimulatory effects on monocytes as well.

Calcium↗

PubMed1991

Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice.

Monoclonal antibodies were raised against a bacterial fusion protein containing amino acids 364 to 623 of the major nonstructural protein, NS-1, of minute virus of mice (MVMp), an autonomous parvovirus. By immunoblot analyses, these antibodies all recognized an 83-kDa protein in MVM-infected mouse fibroblast cells. Indirect immunofluorescence studies showed that five of the six react against a nuclear protein in MVM-infected mouse cells resulting in discrete foci of fluorescence. These foci do not correspond with the nucleoli, the site of MVM DNA replication. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen and showed that four distinct epitopes were recognized by the different antibodies. A 25-amino-acid peptide was used in competition ELISAs to confirm the location of the epitope recognized by two antibodies CE10 and AC6. Preliminary characterization of an NS-1/NS-2 fusion protein synthesized in insect cells using a baculovirus expression vector showed that this fusion protein is also localized within the nucleus; however, in contrast, the full-length NS-1 polypeptide is located within the cytoplasm.

Amino Acid Sequence↗

PubMed1991

Antibodies to interleukin 3 as probes for the interaction of interleukin 3 with its receptor.

A series of antibodies, directed against murine interleukin-3 (IL-3) or synthetic peptides corresponding to portions of the IL-3 sequence, has been used to detect receptor-bound IL-3 on the surface of cells. An assay was developed in which the bound primary antibody was detected using a biotinylated secondary antibody and fluorescein isothiocyanate-labeled streptavidin, followed by analysis on a fluorescence-activated cell sorter. The fluorescence signal was shown to be specific for cells known to express IL-3 receptors and was dependent on the preincubation of cells with IL-3 under conditions that did not allow internalization of receptors. Antisera raised against full-length synthetic IL-3 or to the N-terminal 29 residues were found to give equivalent signals. On the other hand, antibodies to residues 91-118 showed no signal in this assay, despite being able to bind to IL-3 in solution and neutralize IL-3 bioactivity. When peptides corresponding to residues 30-43 and 123-140 were incubated with the anti-IL-3 antiserum, the majority of the fluorescence signal was abolished, indicating that these two peptides contained the primary epitopes being recognized by the antiserum in this assay. This antiserum also bound to the 91-118 peptide, but the corresponding peptide was not able to reduce the fluorescence signal in a similar competition assay. These results suggest that the 91-118 region is not accessible to antibody when IL-3 is bound to its receptor, whereas at least portions of epitopes 1-29, 30-43, and 123-140 remain accessible to antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1991

Interleukin-8 induces neutrophil transendothelial migration.

Interleukin-8 (IL-8) is a potent neutrophil chemotactic stimulant. We have used chemically synthesized IL-8 to investigate its role in human neutrophil adhesion and transendothelial migration. IL-8 enhanced the adhesiveness of human neutrophils to plastic, and to both unstimulated and tumour necrosis factor (TNF)-stimulated endothelial monolayers in vitro. Using a two-compartment model separated by a confluent endothelial monolayer, we have shown that IL-8 chemotactic stimulation induced transmigration across the monolayer of up to 87.4 +/- 2.1% of added neutrophils (compared to random unstimulated transmigration of 2.2 +/- 0.7%), while chemokinetic stimulation led to transmigration of 21 +/- 3.8% of neutrophils. Preincubation of endothelium with TNF also induced transmigration in this model, and was additive when combined with an IL-8 chemotactic stimulus. Endothelial permeability was increased at maximal rates of chemotactic transmigration, which may correlate with increased permeability of vessels at inflammatory sites in vivo. The property of IL-8 to stimulate movement of neutrophils across endothelial monolayers in vitro supports the concept of a central role for this molecule in the accumulation of neutrophils at inflammatory lesions in vivo.

Capillary Permeability↗

PubMed1990

Interleukin-3 and granulocyte-macrophage colony-stimulating factor mediate rapid phosphorylation and activation of cytosolic c-raf.

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor induce the rapid phosphorylation of the c-raf protein in the growth factor-dependent FDC-P1 and DA-3 murine myeloid cell lines. Furthermore, immunoprecipitates of c-raf isolated from growth factor-stimulated cells demonstrate a marked increase in intrinsic protein kinase activity as measured in vitro. IL-3 and granulocyte-macrophage colony-stimulating factor induce phosphorylation of c-raf at both serine and tyrosine residues. Antiphosphotyrosine immunoprecipitates from IL-3-stimulated cells demonstrate the rapid and coordinate phosphorylation of both c-raf and a protein co-migrating with the 140-kDa putative IL-3 receptor component. Collectively, the findings of rapid and coordinate ligand-induced phosphorylation of a potential IL-3 growth factor receptor component and cytoplasmic c-raf with concomitant c-raf activation provide a cogent sequential molecular model for linking external growth stimuli to intracellular signal transduction events.

Animals↗

PubMed1990

IL-3 promotes production of IL-4 by splenic non-B, non-T cells in response to Fc receptor cross-linkage.

A spleen cell population that lacks CD3, CD4, CD8, Thy-1, B220, and class II major histocompatibility complex cell-surface markers (non-B, non-T cells) produces IL-4 when cultured in wells coated with IgE. Their production of IL-4 in response to plate-bound (PB)-IgE is strikingly enhanced by IL-3, and in the presence of IL-3, these cells also produce IL-4 in response to PB-IgG2a. The effect of IL-3 is not mimicked by IL-1, IL-2, IL-5, IL-6, IL-7, granulocyte-macrophage CSF (GM-CSF) or IFN-gamma. Non-B, non-T cells cultured with IL-3 for 12 h acquire the capacity to produce enhanced amounts of IL-4 in response to subsequent culture with PB-Ig even if IL-3 is omitted from the second culture. Irradiated cells also respond to IL-3 with enhanced capacity to produce IL-4 to PB-Ig, indicating that cell proliferation is not required for the effect of IL-3. The IL-3 effect can be obtained in vivo; treatment of mice with a total dose 90,000 U of synthetic IL-3 over a 3-day period results in the presence of splenic and peritoneal cavity non-B, non-T cells that produce enhanced amounts of IL-4 in response to PB-Ig. The FcR that mediates the response to PB-IgE appears to be Fc epsilon RI because cells can be sensitized with IgE anti-DNP mAb, washed, cultured for 15 h at 37 degrees C, washed again, and stimulated to produce IL-4 with 0.1 to 1 ng/ml of TNP10-OVA. IL-3 does not appear to mediate its function by increasing the number of Fc epsilon RI because it can exert its effect when cultured with non-B, non-T cells after they have been sensitized with IgE anti-DNP. However, IL-3 pretreatment does affect the signaling process in that non-B, non-T cells sensitized with IgE anti-DNP show strikingly reduced production of IL-4 to concentrations of TNP10-OVA of 100 ng/ml or more whereas cells pretreated with IL-3 show little or no diminution in IL-4 production at concentrations of TNP10-OVA up to 1 microgram/ml.

Animals↗

PubMed1990

Neutrophil-activating properties of the melanoma growth-stimulatory activity.

Melanoma growth-stimulatory activity (MGSA), a peptide reported to be mitogenic for Hs294T human melanoma cells, has extensive sequence similarity to the neutrophil-activating peptide NAP-1/IL-8, suggesting functional similarities. To test this hypothesis, MGSA was chemically synthesized and tested for its effects on human neutrophils. It was found to induce chemotaxis, exocytosis of elastase, and changes in cytosolic-free calcium to an extent and at concentrations similar to NAP-1/IL-8. However, MGSA was considerably less potent than NAP-1/IL-8 in inducing the respiratory burst. Intradermal injections in rats of MGSA resulted in a massive accumulation of neutrophils. Our data demonstrate that, apart from its growth-stimulatory activity, MGSA is a potent inflammatory agonist with neutrophil-stimulating properties.

Amino Acid Sequence↗

PubMed1990

Heterogeneity of recombinant granulocyte-macrophage colony-stimulating factor-mediated enhancement of neutrophil adherence to endothelium.

Human recombinant (r) and chemically synthesized granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to enhance the attachment of neutrophils to monolayers of human umbilical vein endothelial cells by direct action upon the neutrophil. Using synthetic peptides of GM-CSF with truncated amino and carboxy termini, a region between amino acids 14 and 24 was found to be essential for neutrophil attachment. In analysis of the response of neutrophils from individual donors, a heterogeneity in their capacity to respond to GM-CSF by increased adherence was observed. The level of response to GM-CSF did not depend on receptor number. However, a positive correlation (r = 0.58) was found between the ability to respond to GM-CSF and the level of response to tumor necrosis factor--suggesting a link between the responses of neutrophils to these two cytokines. The stimulation of neutrophil adhesiveness to endothelial cells by rGM-CSF and the heterogeneity in donor response may have important implications for the clinical administration of GM-CSF.

Adult↗

PubMed1989

Mapping of a putative surface-binding site of human coagulation factor XII.

We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.

Antibodies, Monoclonal↗

PubMed1989

Sandwich enzyme immunoassay for murine IL-3.

A reproducible, sensitive immunoassay for murine interleukin-3 (IL-3) has been developed using two preparations of polyclonal antipeptide antibodies. Rabbits were immunized with the N-terminal peptide 1-29 (IL-3) coupled to KLH and the antibodies were affinity purified on immobilized peptide 1-29 (IL-3). This antibody preparation showed good reactivity with native IL-3, and was used to coat polyvinyl microtiter trays. IL-3 captured by this first antibody was detected by the addition of anti-IL-3 serum (second antibody) raised in sheep against synthetic full length IL-3 (1-140). This test reliably detects IL-3 from every source tested (T cells, WEHI-3B cells, recombinant material from transfected COS 7 cells or murine myeloid FDC-P1 cells transfected with an IL-3 containing retrovirus) with a sensitivity to 2 to 4 U/ml of bioactive IL-3 or about 60 pg synthetic IL-3/ml. The test is performed within 5 to 6 h compared to 2 to 3 d of a standard bioassay.

Animals↗