Novel group of virulence activators within the AraC family that are not restricted to upstream binding sites.

Several regulators within the AraC family control the expression of genes known or thought to be required for virulence of bacterial pathogens. One of these, Rns, activates transcription from an unprecedented variety of binding-site locations. Although nearly all prokaryotic activators bind within a small region upstream and adjacent to the promoter that they regulate, Rns does not bind within this region to activate its own promoter, Prns. Instead, to activate Prns, Rns requires one binding site 224.5 bp upstream and one downstream of the transcription start site. We show in this study that several other AraC family activators recognize the same binding sites as Rns and share with it the ability to utilize a downstream binding site. Like Rns, other members of this group of activators positively regulate the expression of virulence factors in pathogenic bacteria. These regulators are also able to activate transcription from promoter-proximal upstream binding sites since they are able to substitute for Rns at Pcoo, a promoter with only upstream binding sites. Thus, Rns is the prototype for a group of regulators, which include CfaR, VirF, AggR, and CsvR and which activate transcription from locations that are more diverse than those of any other known activator.

SDS-stable complex formation between native apolipoprotein E3 and beta-amyloid peptides.

Extracellular senile plaques composed predominantly of fibrillar amyloid-beta (Abeta) are a major neuropathological feature of Alzheimer's disease (AD). Genetic evidence and in vivo studies suggest that apolipoprotein E (apoE) may contribute to amyloid clearance and/or deposition. In vitro studies demonstrate that native apoE2 and E3 form an SDS-stable complex with Abeta(1-40), while apoE4 forms little such complex. Our current work extends these observations by presenting evidence that apoE3 also binds to Abeta(1-42) and with less avidity to modified species of the peptide found in senile plaque cores. These modified peptides include a form that originates at residue 3-Glu as pyroglutamyl and another with isomerization at the 1-Asp and 7-Asp positions. In addition, we used binding reactions between apoE3 and various Abeta fragments, as well as binding reactions with apoE3 and Abeta(1-40) plus Abeta fragments as competitors, to identify the domain(s) of Abeta involved in the formation of an SDS-stable complex with apoE3. Residues 13-28 of Abeta appear to be necessary, while complex formation is further enhanced by the presence of residues at the C-terminus of the peptide. These results contribute to our understanding of the biochemical basis for the SDS-stable apoE3/Abeta complex and support the hypothesis that Abeta can be transported in vivo complexed with apoE. This complex may then be cleared from the interstitial space by apoE receptors in the brain or become part of an extracellular amyloid deposit.

Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12.

Using a genetic screen we have identified two chromosomal genes, cusRS (ylcA ybcZ), from Escherichia coli K-12 that encode a two-component, signal transduction system that is responsive to copper ions. This regulatory system is required for copper-induced expression of pcoE, a plasmid-borne gene from the E. coli copper resistance operon pco. The closest homologs of CusR and CusS are plasmid-borne two-component systems that are also involved in metal responsive gene regulation: PcoR and PcoS from the pco operon of E. coli; CopR and CopS from the cop operon, which provides copper resistance to Pseudomonas syringae; and SilR and SilS from the sil locus, which provides silver ion resistance to Salmonella enterica serovar Typhimurium. The genes cusRS are also required for the copper-dependent expression of at least one chromosomal gene, designated cusC (ylcB), which is allelic to the recently identified virulence gene ibeB in E. coli K1. The cus locus may comprise a copper ion efflux system, because the expression of cusC is induced by high concentrations of copper ions. Furthermore, the translation products of cusC and additional downstream genes are homologous to known metal ion antiporters.

Rns, a virulence regulator within the AraC family, requires binding sites upstream and downstream of its own promoter to function as an activator.

Strains of enterotoxigenic Escherichia coli that express CS1 and CS2 pili require the transcriptional activator Rns, a member of the AraC family, for the expression of the pilin genes. Rns is also an activator of its own expression. However, the arrangement of its binding sites near its own promoter is unusual for a prokaryotic activator. Most activators have at least one binding site 30-80 nucleotides upstream of the transcription start site, but Rns has a single upstream binding site centred at -227. Rns also has two binding sites downstream of the transcription start site centred at +43 and +82, a region generally thought to be reserved for repressors. In vitro, the binding of a MBP::Rns fusion protein to each of these sites facilitates the binding of RNA polymerase to the rns promoter and the formation of an open complex. In vivo, the upstream binding site and one downstream site are required for Rns-dependent activation of its promoter despite the atypical location of these binding sites for an activator. This suggests that Rns may represent a new class of prokaryotic activators.

Unique lipoproteins secreted by primary astrocytes from wild type, apoE (-/-), and human apoE transgenic mice.

Composition of central nervous system lipoproteins affects the metabolism of lipoprotein constituents within the brain. The epsilon4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer's disease via an unknown mechanism(s). As glia are the primary central nervous system cell type that synthesize apoE, we characterized lipoproteins secreted by astrocytes from wild type (WT), apoE (-/-), and apoE transgenic mice expressing human apoE3 or apoE4 in a mouse apoE (-/-) background. Nondenaturing size exclusion chromatography demonstrates that WT, apoE3, and apoE4 astrocytes secrete particles the size of plasma high density lipoprotein (HDL) composed of phospholipid, free cholesterol, and protein, primarily apoE and apoJ. However, the lipid:apoE ratio of particles containing human apoE is significantly lower than WT. ApoE localizes across HDL-like particle sizes. ApoJ localizes to the smallest HDL-like particles. ApoE (-/-) astrocytes secrete little phospholipid or free cholesterol despite comparable apoJ expression, suggesting that apoE is required for normal secretion of astrocyte lipoproteins. Further, particles were not detected in apoE (-/-) samples by electron microscopy. Nondenaturing immunoprecipitation experiments indicate that apoE and apoJ reside predominantly on distinct particles. These studies suggest that apoE expression influences the unique structure of astrocyte lipoproteins, a process further modified by apoE species.

A conserved residue in the tip proteins of CS1 and CFA/I pili of enterotoxigenic Escherichia coli that is essential for adherence.

Enterotoxigenic Escherichia coli associated with human diarrheal disease utilize any of a limited group of serologically distinguishable pili for attachment to intestinal cells. These include CS1 and CFA/I pili. We show here that chemical modification of arginyl residues in CS1 pili abolishes CS1-mediated agglutination of bovine erythrocytes, which serves as a model system for attachment. Alanine substitution of the single arginyl residue in CooA, the major pilin, had no effect on the assembly of pili or on hemagglutination. In contrast, substitution of alanine for R181 in CooD, the minor pilin associated with the pilus tip, abolished hemagglutination, and substitution of R20 reduced hemagglutination. Neither of these substitutions affected CS1 pilus assembly. This shows that CooD is essential for CS1-mediated attachment and identifies specific residues that are involved in receptor binding but not in pilus assembly. In addition to mediating agglutination of bovine erythrocytes, CFA/I also mediates agglutination of human erythrocytes. Substitution of R181 by alanine in the CooD homolog, CfaE, abolished both of these reactions. We conclude that the same region of the pilus tip protein is involved in adherence of CS1 and CFA/I pili, although their receptor specificities differ. This suggests that the region of the pilus tip adhesin protein that includes R181 might be an appropriate target for therapeutic intervention or for a vaccine to protect against human diarrhea caused by enterotoxigenic E. coli strains that have serologically different pili.

Binding site recognition by Rns, a virulence regulator in the AraC family.

The expression of CS1 pili by enterotoxigenic strains of Escherichia coli is regulated at the transcriptional level and requires the virulence regulator Rns, a member of the AraC family of regulatory proteins. Rns binds at two separate sites upstream of Pcoo (the promoter of CS1 pilin genes), which were identified in vitro with an MBP::Rns fusion protein in gel mobility and DNase I footprinting assays. At each site, Rns recognizes asymmetric nucleotide sequences in two regions of the major groove and binds along one face of the DNA helix. Both binding sites are required for activation of Pcoo in vivo, because mutagenesis of either site significantly reduced the level of expression from this promoter. Thus, Rns regulates the expression of CS1 pilin genes directly, not via a regulatory cascade. Analysis of Rns-nucleotide interactions at each site suggests that binding sites for Rns and related virulence regulators are not easily identified because they do not bind palindromic or repeated sequences. A strategy to identify asymmetric binding sites is presented and applied to locate potential binding sites upstream of other genes that Rns can activate, including those encoding the CS2 and CFA/I pili of enterotoxigenic E. coli and the global regulator virB of Shigella flexneri.

Potassium transport by amino acid permeases in Saccharomyces cerevisiae.

Deletion of the potassium transporter genes TRK1 and TRK2 impairs potassium uptake in Saccharomyces cerevisiae, resulting in a greatly increased requirement for the ion and the inability to grow on low pH medium. Selection for mutations that restored growth of trk1Delta trk2Delta cells on low pH (3.0) medium led to the isolation of a dominant suppressor that also partially suppressed the increased K+ requirement of these cells. Molecular analysis revealed the suppressor to be an allele of BAP2 that encodes a permease for branched chain amino acids. The suppressor mutation (BAP2-1) converts a phenylalanine codon, highly conserved among the amino acid permease genes, to a serine codon in a region predicted to lie within the sixth membrane-spanning domain. Generation of the analogous mutation in the histidine permease produced an allele, HIP1-293, that similarly suppressed the low pH sensitivity of trk1Delta trk2Delta cells. Suppression of trk1Delta trk2Delta phenotypes by BAP2-1 or HIP1-293 was correlated with increased Rb+ uptake. The presence of the substrate amino acids enhanced but was not essential for suppression of trk1Delta trk2Delta phenotypes and increased Rb+ uptake. The conserved site altered by the suppressor mutations appears to be important; his4 HIP1-293 cells show an increased requirement for histidine compared with his4 HIP1 cells.

Differential effect of ionizing radiation on transcription in repair-deficient and repair-proficient mice.

Experiments were designed to examine in vivo changes in total transcription and in the expression of the c-fos gene following whole-body exposure of mice to JANUS fission-spectrum neutrons. Radiation repair-deficient (wst/wst) and -proficient (wst/., C57BL/6 x C3H F1) mice were exposed to JANUS fission-spectrum neutrons calibrated to deliver a gut dose of 50 cGy. Animals were sacrificed less than 10 or at 60 min postirradiation, and gut tissues were removed for study. Our results revealed that, in repair-proficient mice, an immediate depression (relative to untreated control) in total transcription was evident that continued through 1 h postirradiation. Conversely, radiation-sensitive wst/wst mice displayed doubled transcription levels postirradiation. Expression of c-fos was consistently depressed following radiation exposure in control and wst/wst mice. However, the depression of c-fos mRNA was delayed in wst/wst mice relative to controls. These results demonstrate abnormal regulation of transcription and of c-fos mRNA accumulation in repair-deficient wasted mice following exposure to ionizing radiation. In addition, this work documents rapid total transcriptional depression in normal mice following radiation exposure.

Stabilization of thoracic and thoracolumbar fracture-dislocations with Harrington rods and sublaminar wires.

Seventeen unstable thoracic and thoracolumbar fracture-dislocations have been treated by Harrington double-distraction rod technique supplemented by doubled 18-gauge stainless-steel wires applied to two intact laminae above and below the injury. Fourteen of the 17 cases were grossly comminuted and translated beyond 50% of the width of the spinal column. No external bracing was used in the 13 patients with neurologic deficits. At the time of follow-up examinations (greater than 22 months in all patients) there was no loss of reduction or fixation in any patient. Solid fusion was achieved in all patients.

Experimental evaluation of Harrington rod fixation supplemented with sublaminar wires in stabilizing thoracolumbar fracture-dislocations.

Segments of the spinal column of calves were instrumented with Harrington rods with and without sublaminar wires. Grossly unstable lesions were created by resecting a 2-cm segment from the central spinal segment. Testing on an Instron tester documented the improvement in stability provided by the sublaminar wires in this grossly unstable experimental fracture. Improved stability was seen in resisting axial loading, lateral bending, and forward flexion loading. Rotational stability was particularly improved.

Percutaneous pinning of distal radius fractures.

Reduction of distal radius fractures is often difficult to maintain with plaster casting alone. Achieving this goal with an easily executed technique which carries a low morbidity is desirable, especially in multiply injured patients. Between July 1977 and September 1980, 24 high-energy fractures of the distal radius in 22 patients were treated by percutaneous pinning of the radial styloid fragment to the opposite radial cortex. Roentgenograms at the time of pinning and 8 weeks or more in followup demonstrating healing were available in 20 patients (22 fractures). Using a previously described method of roentgenographic grading, these cases were evaluated. At followup five fractures were felt to anatomically excellent, 16 good, and one poor. Five fractures in review were felt to be not ideally pinned and these represented three of the five fractures that lost a grade of evaluation rating during healing. One superficial pin tract infection developed and two patients complained of local pin discomfort. These complications resolved with pin removal. If guidelines for pinning are followed properly, percutaneous pinning provides a simple and useful method for maintaining reduction in unstable distal radius fractures in multiply injured patients.