scholarly journals Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility

1992 ◽  
Vol 117 (6) ◽  
pp. 1343-1350 ◽  
Author(s):  
C Hardwick ◽  
K Hoare ◽  
R Owens ◽  
HP Hohn ◽  
M Hook ◽  
...  
Keyword(s):  
Cdna Expression Library ◽  
Receptor Complex ◽  
Ras Gene ◽  
Expression Library ◽  
Hybrid Gene ◽  
Cell Locomotion ◽  
Tumor Cell Motility ◽  
Metallothionein Promoter ◽  
New Type ◽  
Hyaluronan Receptor

A cDNA encoding a unique hyaluronan receptor has been molecularly cloned from a lambda GT11 3T3 cDNA expression library. Immunoblot analyses of cell lysates, using antibodies to peptides encoded in the cDNA, specifically react with a 58-kD protein. This protein is regulated by the mutant H-ras gene in cells containing a metallothionein promoter H-ras hybrid gene. Further, antibodies to peptide sequences encoded in the cDNA block the increase in locomotion resulting from induction of the mutant H-ras gene in this cell line. In a transblot assay, the bacterially expressed protein binds to biotinylated hyaluronan. Antibodies to peptides encoded in the cDNA react in immunoblot assays with the 58- and 52-kD proteins of a novel hyaluronan receptor complex previously implicated in cell locomotion. Furthermore, antibodies specific to the 58- and 52-kD proteins, which block ras-induced locomotion, also cross-react with the expressed, encoded protein. The gene product described here appears to be a new type of hyaluronan receptor that is involved in cell locomotion. It is named RHAMM, an acronym for receptor for hyaluronan-mediated motility.

scholarly journals Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells.

1991 ◽  
Vol 112 (5) ◽  
pp. 1041-1047 ◽  
Author(s):  
E A Turley ◽  
L Austen ◽  
K Vandeligt ◽  
C Clary
Keyword(s):  
Binding Sites ◽  
Binding Protein ◽  
Experimental Period ◽  
Cell Binding ◽  
Ras Gene ◽  
Hybrid Gene ◽  
Cell Locomotion ◽  
A Cell ◽  
Dose Dependent ◽  
Hyaluronan Binding

Hyaluronan (HA) and one of its cell binding sites, fibroblast hyaluronan binding protein (HABP), is shown to contribute to the regulation of 10T1/2 cell locomotion that contain an EJ-ras-metallothionein (MT-1) hybrid gene. Promotion of the ras-hybrid gene with zinc sulfate acutely stimulates, by 6-10-fold, cell locomotion. After 10 h, locomotion drops to two- to threefold above that of uninduced cells. Several observations indicate increased locomotion is partly regulated by HA. These include the ability of a peptide that specifically binds HA (HABR) to reduce locomotion, the ability of HA (0.001-0.1 micrograms/ml), added at 10-30 h after induction to stimulate locomotion back to the original, acute rate, and the ability of an mAb specific to a 56-kD fibroblast HABP to block locomotion. Further, both HA and HABP products are regulated by induction of the ras gene. The effect of exogenous HA is blocked by HABR, is dose-dependent and specific in that chondroitin sulfate or heparan have no significant effect. Stimulatory activity is retained by purified HA and lost upon digestion with Streptomyces hyaluronidase indicating that the activity of HA resides in its glycosaminoglycan chain. Uninduced cells are not affected by HA, HABR, or mAb and production of HA or HABP is not altered during the experimental period. These results suggest that ras-transformation activates an HA/HABP locomotory mechanism that forms part of an autocrine motility mechanism. Reliance of induced cells on HA/HABP for locomotion is transient and specific to the induced state.

Isolation of a novel ras gene from Trichomonas vaginalis: a possible evolutionary ancestor of the Ras and Rap genes of higher eukaryotesThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

2007 ◽  
Vol 85 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Ming-yan Xu ◽  
Ju-li Liu ◽  
Ren-li Zhang ◽  
Yu-cai Fu
Keyword(s):  
Signal Transduction ◽  
Trichomonas Vaginalis ◽  
Peer Review Process ◽  
Signal Transduction Pathways ◽  
Cdna Expression Library ◽  
Ras Gene ◽  
Expression Library ◽  
Ancestral Gene ◽  
Intronless Gene ◽  
Divergence Point

The Ras subfamily proteins are small, monomeric GTP-binding proteins with vital roles in regulating eukaryotic signal transduction pathways. Gene duplication and divergence have been postulated as the mechanism by which such family members have evolved their specific functions. A cDNA clone of TvRsp was isolated and sequenced from a cDNA expression library of the primitive eukaryote Trichomonas vaginalis . The genomic DNA corresponding to the cDNA sequence was amplified by PCR and sequenced. Sequence analysis suggested that TvRsp was an intronless gene. This gene encoded a protein of 181 amino acids and contained the 5 conserved G domains that designated it as a Ras or Rap subfamily member. However, the deduced amino acid sequence shared only 34%–37% overall identity with other Ras subfamily members of different species, and the presence of motifs characteristic of both the Ras and Rap families of GTPase confused the familial classification of this gene. Phylogenetic analysis showed its origins at the divergence point of the Ras/Rap families and suggested that TvRsp was a possible evolutionary ancestral gene of the ras/rap genes of higher eukaryotes. This information was of importance not only from the perspective of understanding the evolution and diversity of eukaryotic signal transduction pathways but also in providing a framework by which to understand protein processing in the growth and differentiation of single-celled microorganisms.

Chicken vitellogenin gene-binding protein, a leucine zipper transcription factor that binds to an important control element in the chicken vitellogenin II promoter, is related to rat DBP

1991 ◽  
Vol 11 (10) ◽  
pp. 4863-4875
Author(s):  
S V Iyer ◽  
D L Davis ◽  
S N Seal ◽  
J B Burch
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Binding Protein ◽  
Expression Profiles ◽  
Control Element ◽  
Cdna Expression Library ◽  
Leucine Zippers ◽  
Expression Library ◽  
Positive Elements ◽  
Vitellogenin Gene

We screened a chicken liver cDNA expression library with a probe spanning the distal region of the chicken vitellogenin II (VTGII) gene promoter and isolated clones for a transcription factor that we have named VBP (for vitellogenin gene-binding protein). VBP binds to one of the most important positive elements in the VTGII promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP was deduced from a nearly full length cDNA copy and was found to contain a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. Moreover, VBP is a stable dimer free in solution. A data base search revealed that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control.

scholarly journals Immunogenic and Plasminogen-Binding Surface-Associated α-Enolase of Trichomonas vaginalis

2007 ◽  
Vol 76 (2) ◽  
pp. 523-531 ◽  
Author(s):  
V. Mundodi ◽  
A. S. Kucknoor ◽  
J. F. Alderete
Keyword(s):  
Trichomonas Vaginalis ◽  
Specific Inhibitor ◽  
Aminocaproic Acid ◽  
Glycolytic Enzyme ◽  
Cdna Expression Library ◽  
Dependent Manner ◽  
Expression Library ◽  
Transmitted Infection ◽  
Sexually Transmitted ◽  
Vaginal Epithelial Cells

ABSTRACT Trichomonas vaginalis is a protist that causes the most common human sexually transmitted infection. A T. vaginalis cDNA expression library was screened with pooled sera from patients with trichomoniasis. A highly reactive cDNA clone of 1,428 bp encoded a trichomonad protein of 472 amino acids with sequence identity to α-enolase (tv-eno1). The sequence alignment confirmed the highly conserved nature of the enzyme with 65% to 84% identity among organisms. The expression of tv-eno1 was up-regulated by contact of parasites with vaginal epithelial cells, and this is the first report demonstrating up-regulation by cytoadherence of a plasminogen-binding α-enolase in T. vaginalis. Immunofluorescence with monoclonal antibody of nonpermeabilized trichomonads showed tv-ENO1 on the surface. The recombinant tv-ENO1 was expressed in Escherichia coli as a glutathione S-transferase (GST)::tv-ENO1 fusion protein, which was cleaved using thrombin to obtain affinity-purified recombinant tv-ENO1 protein (tv-rENO1) detectable in immunoblots by sera of patients. Immobilized tv-rENO1 bound human plasminogen in a dose-dependent manner, and plasminogen binding by tv-rENO1 was confirmed in a ligand blot assay. The plasminogen-specific inhibitor ε-aminocaproic acid blocked the tv-rENO1-plasminogen association, indicating that lysines play a role in binding to tv-rENO1. Further, both parasites and tv-rENO1 activate plasminogen to plasmin that is mediated by tissue plasminogen activator. These data indicate that as with other bacterial pathogens, tv-ENO1 is an anchorless, surface-associated glycolytic enzyme of T. vaginalis.

scholarly journals Glyceraldehyde-3-Phosphate Dehydrogenase Is a Surface-Associated, Fibronectin-Binding Protein of Trichomonas vaginalis

2009 ◽  
Vol 77 (7) ◽  
pp. 2703-2711 ◽  
Author(s):  
A. Lama ◽  
A. Kucknoor ◽  
V. Mundodi ◽  
J. F. Alderete
Keyword(s):  
Trichomonas Vaginalis ◽  
Binding Protein ◽  
Transmitted Disease ◽  
Surface Expression ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Sexually Transmitted ◽  
Vaginal Epithelial Cells ◽  
Extracellular Matrix Components ◽  
Fibronectin Binding Protein

ABSTRACT Trichomonas vaginalis colonizes the urogenital tract of humans and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. We have shown an association of T. vaginalis with basement membrane extracellular matrix components, a property which we hypothesize is important for colonization and persistence. In this study, we identify a fibronectin (FN)-binding protein of T. vaginalis. A monoclonal antibody (MAb) from a library of hybridomas that inhibited the binding of T. vaginalis organisms to immobilized FN was identified. The MAb (called ws1) recognized a 39-kDa protein and was used to screen a cDNA expression library of T. vaginalis. A 1,086-bp reactive cDNA clone that encoded a protein of 362 amino acids with identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained. The gapdh gene was cloned, and recombinant GAPDH (rGAPDH) was expressed in Escherichia coli cells. Natural GAPDH and rGAPDH bound to immobilized FN and to plasminogen and collagen but not to laminin. MAb ws1 inhibited binding to FN. GAPDH was detected on the surface of trichomonads and was upregulated in synthesis and surface expression by iron. Higher levels of binding to FN were seen for organisms grown in iron-replete medium than for organisms grown in iron-depleted medium. In addition, decreased synthesis of GAPDH by antisense transfection of T. vaginalis gave lower levels of organisms bound to FN and had no adverse effect on growth kinetics. Finally, GAPDH did not associate with immortalized vaginal epithelial cells (VECs), and neither GAPDH nor MAb ws1 inhibited the adherence of trichomonads to VECs. These results indicate that GAPDH is a surface-associated protein of T. vaginalis with alternative functions.

Identification of 4 new HLA-DR–restricted minor histocompatibility antigens as hematopoietic targets in antitumor immunity

Blood ◽  
2009 ◽  
Vol 114 (17) ◽  
pp. 3684-3692 ◽  
Author(s):  
Anita N. Stumpf ◽  
Edith D. van der Meijden ◽  
Cornelis A. M. van Bergen ◽  
Roel Willemze ◽  
J. H. Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Hematopoietic Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Histocompatibility Antigens ◽  
Minor Histocompatibility Antigens ◽  
Hla Dr

Abstract Potent graft-versus-leukemia (GVL) effects can be mediated by donor-derived T cells recognizing minor histocompatibility antigens (mHags) in patients treated with donor lymphocyte infusion (DLI) for relapsed hematologic malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). Donor-derived T cells, however, may not only induce GVL, but also mediate detrimental graft-versus-host disease (GVHD). Because HLA-class II is under noninflammatory conditions predominantly expressed on hematopoietic cells, CD4+ T cells administered late after alloSCT may selectively confer GVL without GVHD. Although a broad range of different HLA-class I–restricted mHags have been identified, the first 2 autosomal HLA-class II–restricted mHags have only recently been characterized. By screening a recombinant bacteria cDNA expression library, we identified 4 new HLA-class II–restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia who achieved long-term complete remission and experienced only mild GVHD of the skin after DLI. All CD4+ T cells were capable of recognizing the mHags presented by HLA-DR surface molecules on primary hematopoietic cells, but not on skin-derived (cytokine-treated) fibroblasts. The selective recognition of hematopoietic cells as well as the balanced population frequencies and common HLA-DR restriction elements make the novel mHags possible targets for development of immunotherapeutic strategies.

Identification of novel IgE-binding soybean allergens using serological analysis of a recombinant cDNA expression library (SEREX)

2014 ◽  
Vol 23 (4) ◽  
pp. 1037-1042 ◽  
Author(s):  
Jae-Hwan Kim ◽  
Kang-Mo Ahn ◽  
Wooki Kim ◽  
Youngshin Han ◽  
Young-Rok Kim ◽  
...  
Keyword(s):  
Cdna Expression Library ◽  
Serological Analysis ◽  
Expression Library ◽  
Ige Binding ◽  
Cdna Expression
Sign in / Sign up

Export Citation Format

Share Document