Llama single-domain antibodies directed against nonconventional epitopes of tumor-associated carcinoembryonic antigen absent from nonspecific cross-reacting antigen.

Single-domain antibodies (sdAbs), which occur naturally in camelids, are endowed with many characteristics that make them attractive candidates as building blocks to create new antibody-related therapeutic molecules. In this study, we isolated from an immunized llama several high-affinity sdAbs directed against human carcinoembryonic antigen (CEA), a heavily glycosylated tumor-associated molecule expressed in a variety of cancers. These llama sdAbs bind a different epitope from those defined by current murine mAbs, as shown by binding competition experiments using immunofluorescence and surface plasmon resonance.

A llama single domain anti-idiotypic antibody mimicking HER2 as a vaccine: Immunogenicity and efficacy.

HER2 over-expression in breast cancer correlates with reduced survival. Anti-idiotypic antibodies (Abs) that closely mimic HER2 could play a crucial role in the development of effective therapeutic vaccines. Here, we show that an anti-idiotypic single domain antibody (sdAb) 1HE isolated from a library generated from a Trastuzumab F(ab')(2)-immunized llama, closely mimics HER2.

NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: model of the possible lipid A-binding site.

The anti-lipopolysaccharide factor ALF-Pm3 is a 98-residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter as a folded and (15)N uniformly labeled rALF-Pm3 protein. Its 3D structure was established by NMR and consists of three alpha-helices packed against a four-stranded beta-sheet.

A homogeneous resonance energy transfer-based assay to monitor MutS/DNA interactions.

Probing the interactions of the DNA mismatch repair protein MutS with altered and damaged DNA is of great interest both for the understanding of the mismatch repair system function and for the development of tools to detect mutations. Here we describe a homogeneous time-resolved fluorescence (HTRF) assay to study the interactions of Escherichia coli MutS protein with various DNA substrates. First, we designed an indirect HTRF assay on a microtiter plate format and demonstrated its general applicability through the analysis of the interactions between MutS and mismatched DNA or DNA containing the most common lesion of the anticancer drug cisplatin.

Evidence of a bactericidal permeability increasing protein in an invertebrate, the Crassostrea gigas Cg-BPI.

A cDNA sequence with homologies to members of the LPS-binding protein and bactericidal/permeability-increasing protein (BPI) family was identified in the oyster Crassostrea gigas. The recombinant protein was found to bind LPS, to display bactericidal activity against Escherichia coli, and to increase the permeability of the bacterial cytoplasmic membrane. This indicated that it is a BPI rather than an LPS-binding protein.

Structure-activity relationships of phenyl-furanyl-rhodanines as inhibitors of RNA polymerase with antibacterial activity on biofilms.

The dramatic rise of antibiotic-resistant bacteria over the past two decades has stressed the need for completely novel classes of antibacterial agents. Accordingly, recent advances in the study of prokaryotic transcription open new opportunities for such molecules. This paper reports the structure-activity relationships of a series of phenyl-furanyl-rhodanines (PFRs) as antibacterial inhibitors of RNA polymerase (RNAP).

Antibody-antigenic peptide interactions monitored by SPR and QCM-D. A model for SPR detection of IA-2 autoantibodies in human serum.

This work reports on a complementary use of surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D) technologies to study interactions between a peptide antigen and polyclonal antibodies, in an experimental format suitable for diagnostic assays of autoimmune diseases. In the chosen model, a synthetic peptide from the juxtamembrane region of IA-2 (a type 1 diabetes associated antigen) was immobilized by an optimized chemical protocol applicable to both BIACORE and QCM-D sensors. A thorough study of the peptide immobilization was performed to optimize the signal-to-noise ratio using mixed self-assembled monolayers (SAM) on a gold surface.

Generation of llama single-domain antibodies against methotrexate, a prototypical hapten.

Single-domain antibodies specific to methotrexate (MTX) were obtained after immunization of one llama (Llama glama). Specific VHH domains (V-D-J-REGION) were selected by panning from an immune-llama library using phage display technology. The antibody fragments specific to MTX were purified from Escherichia coli (C41 strain) periplasm by immobilized metal affinity chromatography with an expression level of around 10mg/L.

Monoclonal antibody 76F distinguishes IA-2 from IA-2beta and overlaps an autoantibody epitope.

IA-2 and IA-2beta are highly related proteins that are autoantigens in type 1 diabetes, and provide a model for developing reagents and assays that distinguish similar proteins with unique autoantibody epitopes. Monoclonal antibodies (mAb) to IA-2 and IA-2beta were prepared and tested for their ability to bind to the related proteins and their ability to compete for specific autoantibody epitope binding by sera from patients with type 1 diabetes. Monoclonal antibodies that specifically bound IA-2 (76F) or bound both IA-2 and IA-2beta (A9) were isolated and characterized.

Biological activities on T lymphocytes of a baculovirus-expressed chimeric recombinant IgG1 antibody with specificity for the CDR3-like loop on the D1 domain of the CD4 molecule.

A baculovirus-expressed chimeric recombinant IgG1 (rIgG1) antibody, with Cgamma1 and Ckappa human constant domains, was derived from the murine monoclonal antibody (mAb) 13B8.2, which is specific for the CDR3-like loop of the CD4 molecule and which inhibits HIV-1 replication. Chimeric rIgG1 antibody 13B8.

HcPro, a multifunctional protein encoded by a plant RNA virus, targets the 20S proteasome and affects its enzymic activities.

The proteasome is a multicatalytic complex involved in many cellular processes in eukaryotes, such as protein and RNA turnover, cell division, signal transduction, transcription and translation. Intracellular pathogens are targets of its enzymic activities, and a number of animal viruses are known to interfere with these activities. The first evidence that a plant virus protein, the helper component-proteinase (HcPro) of Lettuce mosaic virus (LMV; genus Potyvirus), interferes with the 20S proteasome ribonuclease is reported here.

Highly conserved beta16/beta17 beta-hairpin structure in human immunodeficiency virus type 1 YU2 gp120 is critical for CCR5 binding.

Whereas gp120 CD4-induced structures have been largely documented and at least in part elucidated by crystallization, information about gp120 coreceptor-induced structures remains incomplete despite numerous studies. In this work, mutations were carried out in a selected internal region of HIV-1/YU2 gp120, proximal to the CD4-binding site, because of its highly conserved nature among retroviruses and its high structural stability. The targeted residues, belonging to the beta16/beta17 beta-hairpin, modulate gp120 binding to CD4 and gp120-CD4 complex binding to CCR5.

Changes in the dimeric state of neuronal nitric oxide synthase affect the kinetics of secretagogue-induced insulin response.

We previously showed that pancreatic beta-cells express a neuronal isoform of nitric oxide synthase (nNOS) that controls insulin secretion by exerting two enzymatic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now bring evidence that two inhibitors of nNOS, N-omega-nitro-l-arginine methyl ester (l-NAME) and 7-nitroindazole (7-NI), increase glucose-induced insulin secretion but affect beta-cell function differently. In the presence of l-NAME, insulin response is monophasic, whereas 7-NI preserves the normal biphasic secretory pattern.

Directed mutagenesis in region 713-720 of human thyroperoxidase assigns 713KFPED717 residues as being involved in the B domain of the discontinuous immunodominant region recognized by human autoantibodies.

Autoantibodies (aAbs) to thyroid peroxidase (TPO), the hallmark of autoimmune thyroid disease (AITD), recognize conformational epitopes restricted to an immunodominant region (IDR), divided into two overlapping domains A and B. Despite numerous efforts aimed at localizing the IDR and identifying aAb-interacting residues on TPO, only two critical amino acids, Lys(713) and Tyr(772), have been characterized. Precise and complete delineation of the other residues involved in the IDR remains to be defined.

Peptides derived from the two regulatory domains of p53 are recognized by two p53-activating antibodies.

The C-terminus of the transcription factor p53 seems to play an important role by controlling the specific DNA-binding activity, which is directly associated with sensing damaged DNA. Another region located in the N-terminus of the protein has also been shown to regulate the DNA-binding activity of the protein. This activity can be promoted by peptides derived from these two negative regulatory regions or by binding of antibodies directed against the C-terminus of the p53 protein.

Mapping the paratope of anti-CD4 recombinant Fab 13B8.2 by combining parallel peptide synthesis and site-directed mutagenesis.

We analyzed antigen-binding residues from the variable domains of anti-CD4 antibody 13B8.2 using the Spot method of parallel peptide synthesis. Sixteen amino acids, defined as Spot critical residues (SCR), were identified on the basis of a 50% decrease in CD4 binding to alanine analogs of reactive peptides.

Localization of the discontinuous immunodominant region recognized by human anti-thyroperoxidase autoantibodies in autoimmune thyroid diseases.

The discontinuous immunodominant region (IDR) recognized by autoantibodies directed against the thyroperoxidase (TPO) molecule, a major autoantigen in autoimmune thyroid diseases, has not yet been completely localized. By using peptide phage-displayed technology, we identified three critical motifs, LXPEXD, QSYP, and EX(E/D)PPV, within selected mimotopes which interacted with the human recombinant anti-TPO autoantibody (aAb) T13, derived from an antibody phage-displayed library obtained from thyroid-infiltrating TPO-selected B cells of Graves' disease patients. Mimotope sequence alignment on the TPO molecule, together with the binding analysis of the T13 aAb on TPO mutants expressed by Chinese hamster ovary cells, demonstrated that regions 353-363, 377-386, and 713-720 from the myeloperoxidase-like domain and region 766-775 from the complement control protein-like domain are a part of the IDR recognized by the recombinant aAb T13.

Intracellular distribution of viral gene products regulates a complex mechanism of cauliflower mosaic virus acquisition by its aphid vector.

Interactions between Cauliflower mosaic virus (CaMV) and its aphid vector are regulated by the viral protein P2, which binds to the aphid stylets, and protein P3, which bridges P2 and virions. By using baculovirus expression of P2 and P3, electron microscopy, surface plasmon resonance, affinity chromatography, and transmission assays, we demonstrate that P3 must be previously bound to virions in order that attachment to P2 will allow aphid transmission of CaMV. We also show that a P2:P3 complex exists in the absence of virions but is nonfunctional in transmission.