ABSTRACT
Active and passive immunotherapy targeted at the amyloid-beta (Abeta) peptide has been proposed as therapeutic approach against Alzheimer's disease (AD), and efforts towards the generation and application of antibody-based reagents that are capable of preventing and clearing amyloid aggregates are currently under active investigation. Previously, we selected and characterized a new anti-Abeta1-42 phage-displayed scFv antibody, designated clone b4.4, using a non-immune human scFv antibody library and demonstrated that a peptide based on the sequence of the Ig heavy chain (VH) complementarity-determining region (HCDR3) of this antibody fragment bound to Abeta1-42)and had neuroprotective potential against Abeta1-42 mediated neurotoxicity in rat hippocampal cultured neurons. In the present study, using novel computational methods and in vitro experiments we demonstrated that b4.4 binds to the central region of Abeta1-42. We also demonstrated that this scFv antibody binds to Abeta-derived diffusible ligands (ADDLs) and neutralizes the toxicity of both fibrillar and oligomeric forms of Abeta1-42 tested in vitro in SH-SY5Y cell cultures.
Subject(s)
Amyloid beta-Peptides/pharmacology , Complementarity Determining Regions/pharmacology , Immunoglobulin Fragments/pharmacology , Immunoglobulin Heavy Chains/pharmacology , Neuroprotective Agents/pharmacology , Oligopeptides/pharmacology , Peptide Fragments/pharmacology , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/immunology , Cell Line, Tumor , Cell Survival/drug effects , Complementarity Determining Regions/chemistry , Epitope Mapping , Humans , Immunoglobulin Fragments/chemistry , Immunoglobulin Heavy Chains/chemistry , Models, Molecular , Neuroprotective Agents/chemistry , Oligopeptides/chemistry , Peptide Fragments/chemistry , Peptide Fragments/immunology , Protein BindingABSTRACT
The Na(+)-glucose cotransporter (SGLT1) is expressed primarily by small intestinal epithelial cells and transports the monosaccharides glucose and galactose across the apical membrane. Here we describe the isolation and characterization of 5.3 kb of the 5'-flanking region of the SGLT1 gene by transiently transfecting reporter constructs into a variety of epithelial cell lines. A fragment (nt -235 to +22) of the promoter showed strong activity in the intestinal cell line Caco-2 but was inactive in a nonintestinal epithelial cell line (Chinese hamster ovary). Within this region, three cis-elements, a hepatocyte nuclear factor-1 (HNF-1) and two GC box sites are critical for maintaining the gene's basal level of expression. The two GC boxes bind to several members of the Sp1 family of transcription factors and, in the presence of HNF-1, synergistically upregulate transactivation of the promoter. A novel 16-bp element just downstream of one GC box was also shown to influence the interaction of Sp1 to its binding site. In summary, we report the identification and characterization of the human SGLT1 minimal promoter and the critical role that HNF-1 and Sp1-multigene members have in enhancing the basal level of its transcription in Caco-2 cells.
Subject(s)
DNA-Binding Proteins , Gene Expression Regulation/physiology , Membrane Glycoproteins/genetics , Monosaccharide Transport Proteins/genetics , Nuclear Proteins , Sp1 Transcription Factor/physiology , Transcription Factors/physiology , Animals , Base Sequence/genetics , CHO Cells/physiology , Caco-2 Cells/physiology , Cell Line , Cricetinae , DNA Footprinting , Deoxyribonuclease I , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Humans , LLC-PK1 Cells/physiology , Molecular Sequence Data , Multigene Family/physiology , Promoter Regions, Genetic/genetics , Sodium-Glucose Transporter 1 , Sp1 Transcription Factor/metabolism , Swine , TransfectionABSTRACT
The regulatory elements that control basal and activated transcriptional expression of the polymeric IgA receptor gene (pIgR) have not been defined. In this study, we performed functional analysis of the murine pIgR 5'-upstream region. Transient transfection studies identified the gene's minimal promoter to reside within 110 nucleotides upstream from the start of transcription. Substitution mutations of this region identified both a putative activator (-78 to -70) and a repressor (-66 to -52) element. DNase I footprint analysis confirmed an area of protection that spans from nucleotides -85 to -62. Mobility shift assays of the putative region confirmed binding of upstream stimulatory factor 1 (USF1) to an E box element at positions -75 and -70, representing the putative enhancer. Overexpression studies using various forms of USF suggest that both USF1 and USF2 enhance activity of the pIgR minimal promoter. We report the identification and characterization of the murine pIgR minimal promoter, as well as the critical role of USF in enhancing its basal level of transcription in Caco-2 cells.
