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1.
Virus Res ; 163(1): 223-8, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22020362

ABSTRACT

BACKGROUND: The genome of human cytomegalovirus (HCMV) has been studied extensively, particularly in the UL/b' region. In this study, transcripts of one of the UL/b' genes, UL146, were identified in 3 HCMV isolates obtained from urine samples of congenitally infected infants. METHODS: Northern blot hybridization, cDNA library screening, and RACE-PCR were used. RESULTS: All sequences of clones from a cDNA library were about 3225 bp in length with the same 5' and 3' ends. The results were accordant with that analyzed by Northen-blot and RACE-PCR in three HCMV clinical isolates. The transcript initiated from the region upstream of UL146 flanking region and terminated just downstream of UL132 including UL146, UL147, UL147A, UL148, and UL132 ORFs. Treatment of the infected cells with phosphonoacetic acid inhibited its transcription. CONCLUSIONS: UL146 ORF was transcribed with 4 downstream ORFs from UL147 to UL132 at true late kinetics. The transcript of UL146 initiated at 73nt upstream of UL146 and terminated just downstream of UL132 in the 3 clinical isolates.


Subject(s)
Chemokines, CXC/biosynthesis , Chemokines, CXC/genetics , Cytomegalovirus Infections/congenital , RNA, Messenger/genetics , RNA, Viral/isolation & purification , Transcription, Genetic , Urine/virology , Viral Proteins/biosynthesis , Viral Proteins/genetics , Blotting, Northern , Chemokines, CXC/isolation & purification , Cytomegalovirus Infections/virology , DNA, Complementary/genetics , DNA, Complementary/metabolism , Gene Library , Humans , Infant , Molecular Sequence Data , RNA, Messenger/isolation & purification , RNA, Viral/genetics , Sequence Analysis, DNA , Viral Proteins/isolation & purification
2.
Appl Biochem Biotechnol ; 164(8): 1366-75, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21431320

ABSTRACT

Mouse CXCL14/BRAK is a monocyte-selective chemokine which is expressed in almost all normal tissues. A flood of reports on its new functions of tumor suppression and fat metabolism modulation has left CXCL14 a potential therapeutic candidate for these diseases. Therefore, a simple accessible method is on demand for large-scale production of recombinant mouse CXCL14 protein for in vivo animal studies. Here, we introduce an efficient method for large-scale production of recombinant mouse CXCL14, by which an 18-mg protein is produced from 2-L Escherichia coli culture with good bioactivity and low level of endotoxin.


Subject(s)
Chemokines, CXC/genetics , Chemokines, CXC/isolation & purification , Escherichia coli/genetics , Amino Acid Sequence , Animals , Base Sequence , Chemokines, CXC/metabolism , Chromatography, Affinity , Endotoxins/metabolism , Mice , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
3.
Circulation ; 116(15): 1683-92, 2007 Oct 09.
Article in English | MEDLINE | ID: mdl-17875967

ABSTRACT

BACKGROUND: Local delivery of chemotactic factors represents a novel approach to tissue regeneration. However, successful chemokine protein delivery is challenged by barriers including the rapid diffusion of chemokines and cleavage of chemokines by proteases that are activated in injured tissues. Stromal cell-derived factor-1 (SDF-1) is a well-characterized chemokine for attracting stem cells and thus a strong candidate for promoting regeneration. However, SDF-1 is cleaved by exopeptidases and matrix metalloproteinase-2, generating a neurotoxin implicated in some forms of dementia. METHODS AND RESULTS: We designed a new chemokine called S-SDF-1(S4V) that is resistant to matrix metalloproteinase-2 and exopeptidase cleavage but retains chemotactic bioactivity, reducing the neurotoxic potential of native SDF-1. To deliver S-SDF-1(S4V), we expressed and purified fusion proteins to tether the chemokine to self-assembling peptides, which form nanofibers and allow local delivery. Intramyocardial delivery of S-SDF-1(S4V) after myocardial infarction recruited CXCR4+/c-Kit+ stem cells (46+/-7 to 119+/-18 cells per section) and increased capillary density (from 169+/-42 to 283+/-27 per 1 mm2). Furthermore, in a randomized, blinded study of 176 rats with myocardial infarction, nanofiber delivery of the protease-resistant S-SDF-1(S4V) improved cardiac function (ejection fraction increased from 34.0+/-2.5% to 50.7+/-3.1%), whereas native SDF-1 had no beneficial effects. CONCLUSIONS: The combined advances of a new, protease-resistant SDF-1 and nanofiber-mediated delivery promoted recruitment of stem cells and improved cardiac function after myocardial infarction. These data demonstrate that driving chemotaxis of stem cells by local chemokine delivery is a promising new strategy for tissue regeneration.


Subject(s)
Chemokines, CXC/genetics , Matrix Metalloproteinase 2/metabolism , Myocardial Infarction/physiopathology , Neovascularization, Physiologic , Stem Cells/physiology , Animals , Blood Pressure , Carotid Arteries/physiology , Chemokine CXCL12 , Chemokines, CXC/isolation & purification , Chemokines, CXC/pharmacology , Dipeptidyl Peptidase 4/metabolism , Drug Resistance , Hematopoietic Stem Cell Mobilization , Male , Mutation , Peptide Hydrolases/metabolism , Plasmids , Rats , Rats, Sprague-Dawley , Regeneration
4.
Protein Expr Purif ; 50(2): 203-14, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16962791

ABSTRACT

CXCR4 belongs to the family of G protein-coupled receptors and mediates the various developmental and regulatory effects of the chemokine SDF-1alpha. In addition, CXCR4 acts as a co-receptor along with CD4 for the HIV-1 viral glycoprotein gp120. Recently, there has also been a small molecule described that antagonizes both SDF-1 and gp120 binding to CXCR4. The structural and mechanistic basis for this dual recognition ability of CXCR4 is unknown largely due to the technical challenges of biochemically producing the components of the various complexes. We expressed the human CXCR4 receptor using a modified baculovirus expression vector that facilitates a single step antibody affinity purification of CXCR4 to >80% purity from Hi5 cells. The recombinant receptor undergoes N-linked glycosylation, tyrosine sulfation and is recognized by the 12G5 conformation specific antibody against human CXCR4. We are able to purify CXCR4 alone as well as complexed with its endogenous ligand SDF-1, its viral ligand gp120, and a small molecule antagonist AMD3100 by ion-exchange chromatography. We anticipate that the expression and purification scheme described in this paper will facilitate structure-function studies aimed at elucidating the molecular basis for CXCR4 recognition of its endogenous chemokine and viral ligands.


Subject(s)
CD4 Antigens/metabolism , Chemokines, CXC/metabolism , HIV Envelope Protein gp120/metabolism , Heterocyclic Compounds/metabolism , Receptors, CXCR4/metabolism , Amino Acid Sequence , Baculoviridae/genetics , Base Sequence , Benzylamines , CD4 Antigens/genetics , CD4 Antigens/isolation & purification , Cells, Cultured , Chemokine CXCL12 , Chemokines, CXC/genetics , Chemokines, CXC/isolation & purification , Cyclams , Dimerization , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/isolation & purification , Humans , Molecular Sequence Data , Protein Binding , Protein Processing, Post-Translational , Receptors, CXCR4/genetics , Receptors, CXCR4/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
5.
Curr HIV Res ; 3(4): 361-9, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16250882

ABSTRACT

INTRODUCTION: CC and CXC chemokines may play a role in mother-to-child HIV-1 transmission by blocking HIV-1 binding to chemokine receptors and impeding viral entry into cells. METHODS: To define correlates of breastmilk chemokines and associations with infant HIV-1 acquisition, chemokines in breastmilk and infant HIV-1 infection risk were assessed in an observational, longitudinal cohort study. We measured MIP-1alpha, MIP-1beta, RANTES, and SDF-1 in month 1 breastmilk specimens from HIV-1-infected women in Nairobi and HIV-1 viral load was calculated in maternal plasma and breastmilk at delivery and 1 month postpartum. Infant infection status was determined at birth and months 1, 3, 6, 9, and 12. RESULTS: Among 281 breastfeeding women, 60 (21%) of their infants acquired HIV-1 during follow-up, 39 (65%) of whom became infected intrapartum or after birth. MIP-1alpha, MIP-1beta, RANTES, and SDF-1 were all positively correlated with breastmilk HIV-1 RNA (P<0.0005). Women with clinical mastitis had 50% higher MIP-1alpha and MIP-1beta levels (P<0.001 and P=0.006, respectively) and women with subclinical mastitis (breastmilk Na(+)/K(+)>1) had approximately 70% higher MIP-1alpha, MIP-1beta and RANTES (P<0.002 for all) compared to women without mastitis. Independent of breastmilk HIV-1, increased MIP-1beta and SDF-1 were associated with reduced risk of infant HIV-1 (RR=0.4; 95% CI 0.2-0.9; P=0.03 and RR=0.5; 95% CI=0.3-0.9; P=0.02, respectively) and increased RANTES was associated with higher transmission risk (RR=2.3; 95% CI 1.1- 5.3; P=0.04). CONCLUSIONS: These observations suggest a complex interplay between virus levels, breastmilk chemokines, and mother-to-child HIV-1 transmission and may provide insight into developing novel strategies to reduce infection across mucosal surfaces.


Subject(s)
Chemokines, CC/isolation & purification , Chemokines, CXC/isolation & purification , HIV Infections/transmission , Infectious Disease Transmission, Vertical , Milk, Human/chemistry , Adolescent , Adult , Chemokine CCL3 , Chemokine CCL4 , Chemokine CCL5/analysis , Chemokine CXCL12 , Chemokines, CXC/analysis , Cohort Studies , Female , Humans , Infant, Newborn , Longitudinal Studies , Macrophage Inflammatory Proteins/analysis , RNA, Viral/analysis , Risk Factors
6.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 21(2): 251-4, 2004 Apr.
Article in Chinese | MEDLINE | ID: mdl-15143551

ABSTRACT

A DNA fragment encoding mouse B Lymphocyte Chemoattractant (BLC, MV10Kda) was obtained by PCR. The amplified fragment was inserted into prokaryotic expression vector PQE30. Recombinant protein was expressed in E. Coli XL-1 blue and purified by affinity chromatography on a nickel-nitrilotriacetic acid gel matrix. Then it was identified by sequence analysis and Western blot analysis. The fragment inserted into prokaryotic expression vector PQE30 was identified to be BLC gene fragment by sequence analysis. And a specfic band was shown by Western blot analysis. These findings provide the evidence that the recombinant protein obtained and purified in this study using gene engineering method is mouse B Lymphocyte Chemoattractant.


Subject(s)
Chemokines, CXC/biosynthesis , Animals , Chemokine CXCL13 , Chemokines, CXC/genetics , Chemokines, CXC/isolation & purification , Escherichia coli/genetics , Genetic Vectors , Mice , Mice, Inbred BALB C , Plasmids/genetics , Prokaryotic Cells/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sequence Analysis, DNA
7.
J Leukoc Biol ; 74(5): 880-8, 2003 Nov.
Article in English | MEDLINE | ID: mdl-12960279

ABSTRACT

The chemokine stromal-derived factor-1 (SDF-1) can block human immunodeficiency virus type 1 (HIV-1) infection in vitro by binding to the CXC chemokine receptor, CXCR-4, which serves as a coreceptor for T cell tropic HIV-1. In spite of being constitutively expressed in vivo, SDF-1 does not appear to block HIV-1 infection and spread in vivo. We report that SDF-1 is consistently measured in normal serum (15.4+/-3.0 ng/ml; mean+/-sd) and in serum from AIDS patients (16.6+/-3.7 ng/ml). However, we find that circulating SDF-1 is modified to an inactive form. When exposed to serum, recombinant SDF-1 is specifically and rapidly altered to yield an apparently smaller chemokine that does not bind to SDF-1 receptor-expressing cells, does not have chemoattractive or pre-B cell stimulatory activity, and does not block HIV-1 infection. Thus, serum modification and inactivation contribute to the failure of SDF-1 to block HIV-1 infection and spread in man. The inactivation of circulating SDF-1 may be critical in permitting local gradients to develop and direct cell trafficking.


Subject(s)
Acquired Immunodeficiency Syndrome/prevention & control , Chemokines, CXC/physiology , Endothelium, Vascular/physiology , HIV-1 , Leukocytes, Mononuclear/physiology , Stromal Cells/physiology , Acquired Immunodeficiency Syndrome/blood , Animals , Cell Division , Cell Line , Cells, Cultured , Chemokine CXCL12 , Chemokines, CXC/blood , Chemokines, CXC/immunology , Chemokines, CXC/isolation & purification , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Immunoglobulin G , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/virology , Mice , Receptors, Interleukin-8A/antagonists & inhibitors , Reference Values , Umbilical Veins
8.
Mol Immunol ; 39(13): 829-34, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12617998

ABSTRACT

A novel CXC chemokine was identified for the first time in fish from common carp (Cyprinus carpio L.). The gene was obtained from the head kidney (HK) stimulated with LPS and Con A. The cDNA consists of 619 bp with a 37 bp 5' UTR and a 287 bp 3' UTR. An open reading frame of 368 bp encodes a 97 amino acid peptide, with a putative signal peptide of 20 aa. The gene has four cysteines residues, which are conserved, with first two cysteines separated with phenylalanine. By homology and phylogenetic analysis, the chemokine was found to be closer to human IP-10. Identities were significantly low to the CXC chemokines cloned from lamprey (Lampetra fluviatilis), flounder (Paralichthys olivaceus), rainbow trout (Onchorhynchus mykiss) and zebrafish (Danio rerio). The carp CXC chemokine contains three exons interrupted by two introns. The gene was transcribed from an early time point by stimulation with LPS and Con A. Organs in resting phase as well as stimulated expressed the gene.


Subject(s)
Carps/immunology , Chemokines, CXC/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Carps/genetics , Chemokines, CXC/genetics , DNA, Complementary/genetics , Exons , Introns , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid
10.
Biochem Biophys Res Commun ; 290(2): 865-8, 2002 Jan 18.
Article in English | MEDLINE | ID: mdl-11785982

ABSTRACT

In the presence of thrombopoietin (TPO), megakaryocytes mature by polyploidization and cytoplasmic maturation, and the matured megakaryocytes induce drastic morphological change and proplatelet formation and release a number of platelets. However, the regulatory mechanism of this unique differentiation process is still obscure. We therefore attempted to identify the factors, expression of which is induced by TPO stimulation in mouse bone marrow megakaryocytes. We isolated the mouse homolog of the neutrophil activating peptide-2 (NAP-2). Mouse NAP-2 cDNA encodes a predicted sequence of 113 amino acids and contains the Cys motif (CXC) found in other members of the alpha-chemokine family. At the amino acid level, the predicted mouse NAP-2 has 50.4%, 51.8%, and 72.6% identity with the predicted human, pig, and rat NAP-2, respectively. Northern blot analysis demonstrates that mouse NAP-2 is expressed only in spleen. Furthermore, the RT-PCR technique shows that the mouse NAP-2 gene is clearly upregulated by TPO stimulation in mouse megakaryocytes.


Subject(s)
Chemokines, CXC/isolation & purification , Animals , Base Sequence , Blotting, Northern , COS Cells , Cells, Cultured , Chemokines, CXC/classification , Chemokines, CXC/genetics , Cloning, Molecular , DNA, Complementary/genetics , Megakaryocytes/cytology , Megakaryocytes/drug effects , Megakaryocytes/metabolism , Mice , Molecular Sequence Data , Organ Specificity , Phylogeny , Sequence Homology, Amino Acid , Spleen/cytology , Spleen/metabolism , Thrombopoietin/pharmacology
11.
Eur Cytokine Netw ; 13(4): 462-73, 2002.
Article in English | MEDLINE | ID: mdl-12517732

ABSTRACT

A sequence encoding a CXC - type chemokine from rainbow trout was found to most resemble members of the CXCL9/CXCL10/CXCL11 sub-family. In mammals, all 3 chemokines are regulated by IFN-gamma and are chemotactic for activated T lymphocytes. The trout chemokine (gammaIP1), with a message of 787 nucleotides, contains 100 amino acids in a typical non-ELR CXC chemokine arrangement. A second sequence (gammaIP2), with 6 nucleotide differences in the coding region when compared to the first, was also identified although it is not known whether this is a second functional gene or a second allele. The gene is separated onto 4 exons, and the introns intervene in conserved positions according to the mammalian equivalents. The sequence encoded by the second exon shares the highest amino acid identity (37%) with CXCL10, with lower values of identity to other CXC chemokines (17-31%). Furthermore, phylogenetic analysis groups the trout chemokine with mammalian CXCL9, CXCL10 and CXCL11 peptides. Constitutive expression of gammaIP is seen in trout gill and low level expression in spleen, head kidney and liver. In RTS-11 cells, gammaIP expression can be induced with poly I:C, but not by LPS, suggesting virus-mediated regulation of gammaIP. Intraperitoneal injection of recombinant trout TNF-alpha caused elevation in gammaIP mRNA levels in trout head kidney.


Subject(s)
Chemokines, CXC/genetics , Oncorhynchus mykiss/genetics , Oncorhynchus mykiss/immunology , Amino Acid Sequence , Animals , Base Sequence , Chemokine CXCL10 , Chemokine CXCL11 , Chemokine CXCL9 , Chemokines, CXC/isolation & purification , Cloning, Molecular , DNA, Complementary/genetics , Intercellular Signaling Peptides and Proteins/genetics , Interferon-gamma/physiology , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid
12.
Eur J Biochem ; 268(18): 4992-9, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11559369

ABSTRACT

Chemokines comprise a class of peptides with chemotactic activity towards leukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, post-translational modifications have been shown to affect the effectiveness of chemokines. Although in several studies, natural CXCR3-targeting chemokines have been isolated, detailed information about the proteins and their possible modifications is lacking. Using a combination of liquid chromatography and mass spectrometry we studied the protein profile of CXCR3-targeting chemokines expressed by interferon-gamma-stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more detail using calcium mobilization and chemotaxis assays. We found that the primary structure of human CXCL10 is different from the generally accepted sequence. In addition we identified a C-terminally truncated CXCL10, lacking the last four amino acids. Native CXCL11 was primarily found in its intact mature form but we also found a mass corresponding to an N-terminally truncated human CXCL11, lacking the first two amino acids FP, indicating that this chemokine is a substrate for dipeptidylpeptidase IV. Interestingly, this same truncation was found when we expressed human CXCL11 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expressing CHO cells) as well as its chemotactic activity for CXCR3-expressing T-cells. It is concluded that detailed information on chemokines at the protein level is important to characterize the exact profile of these chemotactic peptides as modifications can severely alter their biological activity.


Subject(s)
Chemokines, CXC/metabolism , Protein Processing, Post-Translational , Receptors, Chemokine/metabolism , Amino Acid Sequence , Animals , CHO Cells , Calcium/metabolism , Calcium Signaling , Cells, Cultured , Chemokine CXCL10 , Chemokine CXCL11 , Chemokines, CXC/chemistry , Chemokines, CXC/isolation & purification , Chemotaxis , Cricetinae , Humans , Interferon-gamma/pharmacology , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/metabolism , Mice , Molecular Sequence Data , Receptors, CXCR3 , Receptors, Chemokine/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Transfection
13.
Protein Expr Purif ; 21(3): 367-77, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11281710

ABSTRACT

Stromal-cell-derived factor-1 (SDF-1alpha) is an 8-kDa chemokine that is constitutively expressed in bone-marrow-derived stromal cells and has been identified as a ligand for the CXCR4 receptor. We produced the chemokine recombinantly as methionine-SDF-1alpha in Escherichia coli without the leader peptide sequence. The protein was denatured, refolded, and further purified by reversed-phase HPLC. SDF-1alpha was shown to be >95% pure as judged by SDS-PAGE. The final yield of purified and refolded SDF-1alpha was 1-2 mg per gram of wet cell paste. The refolded protein is a ligand for the CXCR4 receptor and has been used to block HIV-mediated cell fusion and downmodulates the CXCR4 receptor. Our ability to purify hundreds of milligrams of refolded protein allowed us to conduct detailed studies of the biophysical properties of the protein. We have used a combination of biophysical techniques to study the solution properties of SDF-1alpha. The average mass of SDF-1alpha, as determined by static light scattering, gave us the first indications that the chemokine may self-associate. Further investigation with sedimentation velocity ultracentrifugation confirmed the existence of two species. The measured s(20, W) values defined two masses corresponding to monomer and dimer. Finally, sedimentation equilibrium ultracentrifugation and dynamic light scattering yielded a composite value of 150 +/- 30 microM for the dimerization constant. We conclude that SDF-1alpha exists in a monomer-dimer equilibrium.


Subject(s)
Anti-HIV Agents/chemistry , Anti-HIV Agents/isolation & purification , Chemokines, CXC/chemistry , Chemokines, CXC/isolation & purification , Anti-HIV Agents/metabolism , Chemokine CXCL12 , Chemokines, CXC/genetics , Chemokines, CXC/metabolism , Chromatography, Gel , Chromatography, High Pressure Liquid , Circular Dichroism , Dimerization , Humans , Light , Molecular Weight , Protein Denaturation , Protein Folding , Protein Renaturation , Protein Sorting Signals , Protein Structure, Quaternary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Scattering, Radiation , Sequence Analysis, Protein , Solutions , Thermodynamics , Ultracentrifugation
14.
J Hematother Stem Cell Res ; 10(1): 147-56, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11276368

ABSTRACT

Human recombinant interferon-inducible protein-10 (rIP-10), a C-X-C chemokine, inhibits proliferation of human hematopoietic progenitors responsive to co-stimulation by recombinant steel factor (rSLF), is chemotactic for human monocytes and T-lymphocytes, and promotes T-lymphocyte adhesion to endothelial cells. Because chemokines have four conserved cysteines forming two intramolecular disulfide bridges, we decided to investigate their contribution in the biological activity of rIP-10. Since amino acid residues 22-98 of the sequence predicted by the cDNA constitute the naturally occurring IP-10, they were cloned after an initiating methionine into expression vector pET-3d. Subsequently rIP-10 was purified by enzymatic cell lysis, solubilization of refractile bodies with guanidine hydrochloride, renaturation by dialysis against dilute acetic acid, and sequential ion-exchange and reverse-phase high-performance liquid chromatography. Purified rIP-10 was reduced with 20 mM dithiothreitol, and chemically modified with 100 mM iodoacetamide (IAA), or S-methyl-methanethiosulfonate (MMTS), or N-methylmaleimide (NMM). Radiolabeling experiments demonstrated that 95% of the rIP-10 thiols were modified, and this was confirmed with SDS-PAGE. The biological activity of modified rIP-10 was determined in vitro by inhibition of rSLF-responsive human bone marrow hematopoietic progenitor proliferation and by chemotaxis assays using human T-lymphocytes and monocytes. In both assay systems, the biological activity was evident at rIP-10 concentrations of 20-100 ng/ml. The activity was preserved after modification of rIP-10 by IAA or MMTS, but was abolished after modification by NMM. We conclude that disulfide bridges are not essential for the biological activity of rIP-10.


Subject(s)
Chemokines, CXC/pharmacology , Chemotaxis/drug effects , Hematopoietic Stem Cells/drug effects , Cell Division/drug effects , Chemokine CXCL10 , Chemokines, CXC/isolation & purification , Chemokines, CXC/metabolism , Cloning, Molecular , Disulfides/metabolism , Disulfides/pharmacology , Hematopoietic Stem Cells/cytology , Humans , Monocytes/physiology , Oxidation-Reduction , Recombinant Proteins/pharmacology , T-Lymphocytes/physiology
15.
Biochemistry ; 39(47): 14549-57, 2000 Nov 28.
Article in English | MEDLINE | ID: mdl-11087410

ABSTRACT

The role of interleukin-8 (IL-8) and related CXC chemokines has been demonstrated in many human diseases. However, more profound studies, e.g., by blocking the effect of these inflammatory mediators, request animal models and hence the identification of all human counterparts for commonly used laboratory animals. In this study, we describe the identification of a novel neutrophil chemotactic protein (NCP) of the rabbit. Intact and NH(2)-terminally truncated NCP forms and IL-8 were isolated from LPS-stimulated rabbit alveolar macrophages and purified to homogeneity by a four-step purification procedure. Determination of the complete primary structure of NCP by mass spectrometry and NH(2)-terminal sequencing of natural protein revealed high structural homology with human epithelial cell-derived neutrophil attractant-78 (ENA-78) and granulocyte chemotactic protein-2 (GCP-2), two related ELR(+)CXC chemokines. Intact NCP(1-76) was found to be 10-fold less potent than truncated NCP(7, 8-76) at inducing neutrophil chemotaxis. NCP(7,8-76) was equally potent as intact rabbit IL-8 at chemoattracting human neutrophils and at inducing calcium fluxes in rabbit neutrophils, 1 ng/mL being the minimal effective concentration. However, like IL-8, NCP failed to induce monocyte or eosinophil migration at 300-fold higher concentrations. IL-8 desensitized the calcium increase induced by NCP and vice versa. Finally, intradermal injection of NCP induced a dose-dependent and significant infiltration of neutrophils in mice skin. It can be concluded that NCP is a novel rabbit CXC chemokine that is, like IL-8, implicated in animal models used to study various human disorders in which neutrophils play an important role.


Subject(s)
Chemokines, CXC/isolation & purification , Chemokines, CXC/physiology , Interleukin-8/analogs & derivatives , Macrophages, Alveolar/metabolism , Neutrophils/physiology , Amino Acid Sequence , Animals , Calcium Signaling , Chemokine CXCL5 , Chemokine CXCL6 , Chemokines, CXC/administration & dosage , Chemokines, CXC/biosynthesis , Chemokines, CXC/chemistry , Chemotaxis, Leukocyte , Humans , Inflammation/pathology , Injections, Intradermal , Interleukin-8/administration & dosage , Interleukin-8/chemistry , Interleukin-8/physiology , Macrophage Activation , Molecular Sequence Data , Neutrophils/pathology , Protein Isoforms/administration & dosage , Protein Isoforms/biosynthesis , Protein Isoforms/isolation & purification , Protein Isoforms/physiology , Rabbits , Sequence Analysis, Protein
16.
J Immunol ; 164(10): 5383-8, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10799902

ABSTRACT

IFN-inducible T cell alpha chemoattractant (I-TAC) is a recently discovered member of the CXC chemokine family. It is a potent T cell chemoattractant expressed by IFN-gamma-treated astrocytes, monocytes, keratinocytes, bronchial epithelial cells, and neutrophils. In this study, we show that I-TAC is also expressed by IFN-gamma-treated endothelial cells (EC), both at the mRNA and protein levels. Induction of the I-TAC message is rapid and sustained over 24 h. TNF-alpha does not induce I-TAC mRNA alone, but does act synergistically with IFN-gamma. Blocking Abs to I-TAC, or to its receptor, CXCR3, reduce T cell adhesion to EC monolayers demonstrating that the expressed protein is functional. Finally, the expression of I-TAC by EC is resistant to the immunosuppressive drug cyclosporin A, suggesting that I-TAC may contribute to the chronic immune inflammation characteristic of graft arteriosclerosis.


Subject(s)
Chemokines, CXC/biosynthesis , Interferon-gamma/physiology , T-Lymphocytes/immunology , Capillaries/cytology , Capillaries/immunology , Capillaries/metabolism , Cell Adhesion/immunology , Cell Migration Inhibition , Cell-Free System/immunology , Cell-Free System/metabolism , Cells, Cultured , Chemokine CXCL11 , Chemokines, CXC/genetics , Chemokines, CXC/isolation & purification , Chemotaxis, Leukocyte/immunology , Drug Synergism , Endothelium, Vascular/cytology , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Fibroblasts/immunology , Fibroblasts/metabolism , Humans , RNA, Messenger/biosynthesis , Skin/cytology , Tumor Necrosis Factor-alpha/physiology , Umbilical Veins
17.
J Immunol ; 164(11): 6027-33, 2000 Jun 01.
Article in English | MEDLINE | ID: mdl-10820287

ABSTRACT

Chemokines direct leukocyte recruitment into sites of tissue inflammation and may facilitate recruitment of leukocytes into allografts following transplantation. Although the expression of chemokines during rejection of MHC-disparate allografts has been examined, chemokine expression in MHC-matched/multiple minor histocompatibility Ag-disparate allografts has not been tested. The intraallograft RNA expression of several C-X-C and C-C chemokines was tested during rejection of full thickness skin grafts from B10. D2 donors on control Ig-, anti-CD4 mAb-, and anti-CD8 mAb-treated BALB/c recipients. In all recipients, two patterns of intragraft chemokine expression were observed during rejection of these grafts: 1) macrophage-inflammatory protein-1alpha, macrophage-inflammatory protein-1beta, GRO-alpha (KC), JE, and IFN-gamma-inducible protein (IP-10) were expressed at equivalent levels in allo- and isografts for 2-4 days posttransplant and then returned to low or undetectable levels; and 2) IP-10 and monokine induced by IFN-gamma (Mig) were expressed in the allografts 3 days before rejection was completed, suggesting a possible role in recruiting primed T cells into the allograft. Three days before completion of rejection, intraallograft IP-10 protein was restricted to the epidermis, whereas Mig was located in the lower dermis and associated with the intense infiltration of mononuclear cells. Treatment of B10.D2 recipients with rabbit antiserum to Mig, but not to IP-10, delayed rejection of the allografts 3-4 days. The results suggest that Mig mediates optimal recruitment of T cells into MHC-matched/multiple minor histocompatibility Ag-disparate allografts during rejection.


Subject(s)
Chemokines/biosynthesis , Chemokines/genetics , Graft Rejection/genetics , Graft Rejection/immunology , Intercellular Signaling Peptides and Proteins , Minor Histocompatibility Antigens/genetics , RNA/biosynthesis , Skin Transplantation/immunology , Amino Acid Sequence , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Chemokine CXCL10 , Chemokine CXCL9 , Chemokines, CXC/immunology , Chemokines, CXC/isolation & purification , Chemokines, CXC/metabolism , Female , Gene Expression Regulation/immunology , Histocompatibility Testing , Immune Sera/administration & dosage , Injections, Intraperitoneal , Lymphocyte Depletion , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Molecular Sequence Data , Rabbits , Transplantation, Homologous
18.
J Immunol ; 164(7): 3774-82, 2000 Apr 01.
Article in English | MEDLINE | ID: mdl-10725737

ABSTRACT

SK&F 107647, a previously described synthetic immunomodulatory peptide, indirectly stimulates bone marrow progenitor cells and phagocytic cells, and enhances host defense effector mechanisms in bacterial and fungal infection models in vivo. In vitro, SK&F 107647 induces the production of a soluble mediator that augments colony forming cell (CFU-GM) formation in the presence of CSFs. In this paper we purified and sequenced the stromal cell-derived hematopoietic synergistic factors (HSF) secreted from both murine and human cell lines stimulated with SK&F 107647. Murine HSF is an N-terminal 4-aa truncated form of the CXC chemokine, KC, while human HSF was identified as an N-terminal 4-aa truncated form of the CXC chemokine, GRO beta. In comparison to their full-length forms, truncated KC and truncated GRO beta were 10 million times more potent as synergistic growth stimulants for CFU-GM. Enhanced potency of these novel truncated chemokines relative to their full-length forms was also demonstrated in respiratory burst assays, CD11b Ag expression, and intracellular killing of the opportunistic pathogen, Candida albicans. Administration of truncated KC significantly enhanced survival of mice lethally infected with C. albicans. The results reported herein delineate the biological mechanism of action of SK&F 107647, which functions via the induction of unique specific truncated forms of the chemokines KC and GRO beta. To our knowledge, this represents the first example where any form of KC or GRO beta were purified from marrow stromal cells. Additionally, this is the first demonstration of in vivo efficacy of a CXC chemokine in an animal infectious fungal disease model.


Subject(s)
Antifungal Agents/isolation & purification , Chemokines, CXC/isolation & purification , Chemotactic Factors/isolation & purification , Growth Substances/isolation & purification , Intercellular Signaling Peptides and Proteins , Peptide Fragments/isolation & purification , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/pharmacology , Amino Acid Sequence , Animals , Antifungal Agents/blood , Antifungal Agents/immunology , Bone Marrow Cells/chemistry , Bone Marrow Cells/immunology , Candidiasis/immunology , Candidiasis/mortality , Candidiasis/prevention & control , Cell Line , Chemokine CXCL1 , Chemokines, CXC/blood , Chemokines, CXC/genetics , Chemokines, CXC/immunology , Chemotactic Factors/blood , Chemotactic Factors/genetics , Chemotactic Factors/immunology , Drug Synergism , Female , Growth Substances/blood , Growth Substances/genetics , Growth Substances/immunology , Humans , Immune Sera/pharmacology , Injections, Intraperitoneal , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Neutrophil Activation/immunology , Oligopeptides/administration & dosage , Oligopeptides/pharmacology , Peptide Fragments/blood , Peptide Fragments/genetics , Peptide Fragments/immunology , Recombinant Proteins/chemistry , Stromal Cells/chemistry , Stromal Cells/immunology
19.
J Immunol ; 163(11): 6155-63, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10570306

ABSTRACT

Chemokines are important mediators of leukocyte migration during the inflammatory response. Post-translational modifications affect the biological potency of chemokines. In addition to previously identified NH2-terminally truncated forms, COOH-terminally truncated forms of the CXC chemokine murine granulocyte chemotactic protein-2 (GCP-2) were purified from conditioned medium of stimulated fibroblasts. The truncations generated 28 natural murine GCP-2 isoforms containing 69-92 residues, including most intermediate forms. Both NH2- and COOH-terminal truncations of GCP-2 resulted in enhanced chemotactic potency for human and murine neutrophils in vitro. The truncated isoform GCP-2(9-78) was 30-fold more potent than intact GCP-2(1-92)/LPS-induced CXC chemokine (LIX) at inducing an intracellular calcium increase in human neutrophils. After intradermal injection in mice, GCP-2(9-78) was also more effective than GCP-2(1-92)/LIX at inducing neutrophil infiltration. Similar to human IL-8 and GCP-2, murine GCP-2(9-78) and macrophage inflammatory protein-2 (MIP-2) induced calcium increases in both CXCR1 and CXCR2 transfectants. Murine GCP-2(9-78) could desensitize the calcium response induced by MIP-2 in human neutrophils and vice versa. Furthermore, MIP-2 and truncated GCP-2(9-78), but not intact GCP-2(1-92)/LIX, partially desensitized the calcium response to human IL-8 in human neutrophils. Taken together, these findings point to an important role of post-translationally modified GCP-2 to replace IL-8 in the mouse.


Subject(s)
Chemokines, CXC/pharmacology , Chemotaxis, Leukocyte , Neutrophils/drug effects , Amino Acid Sequence , Animals , Antigens, CD/genetics , Calcium Signaling , Chemokine CXCL2 , Chemokine CXCL6 , Chemokines, CXC/isolation & purification , Humans , Mice , Molecular Sequence Data , Monokines/pharmacology , Neutrophil Infiltration , Peptide Fragments , Protein Isoforms/isolation & purification , Protein Isoforms/pharmacology , Receptors, Chemokine/genetics , Receptors, Interleukin/genetics , Receptors, Interleukin-8A , Receptors, Interleukin-8B , Sequence Alignment , Sequence Deletion , Species Specificity
20.
J Chromatogr B Biomed Sci Appl ; 729(1-2): 369-74, 1999 Jun 11.
Article in English | MEDLINE | ID: mdl-10410964

ABSTRACT

Chemokines are members of the chemotactic cytokines family implicated in various immunoregulatory functions. The CXC-chemokine stromal cell derived factor-1 (SDF-1alpha) was purified from the culture medium of murine bone marrow stromal cell line (MS-5) by affinity and reversed-phase liquid chromatography. Yield and purity were assessed by capillary electrophoresis (CE) with reference to the human SDF-1alpha from recombinant DNA technology. CE technique was useful to evaluate the purity of human SDF-1alpha from chemical synthesis and to resolve murine and human SDF-1alpha, differing by only one amino acid. Chemotactic activity of the murine SDF-1alpha was tested on the basis of CE quantification.


Subject(s)
Chemokines, CXC/isolation & purification , Chromatography, High Pressure Liquid/methods , Electrophoresis, Capillary/methods , Animals , Cell Line , Chemokine CXCL12 , Chemotaxis, Leukocyte , Humans , Mice
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