"Peptabody": a new type of high avidity binding protein.

A new type of high avidity binding molecule, termed "peptabody" was created by harnessing the effect of multivalent interaction. A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1 surface Ig idiotype, was selected from a phage display library. A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain. The Pab-S fusion protein was expressed in Escherichia coli in a soluble form at high levels and purified in a single step by metal-affinity chromatography. Pab-S specifically bound the BCL1 surface idiotype with an avidity of about 1 nM, which corresponds to a 2 x 10(5)-fold increase compared with the affinity of the synthetic peptide S itself. Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds. Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity. This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties.

Polarity and specific sequence requirements of peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor heterodimer binding to DNA. A functional analysis of the malic enzyme gene PPAR response element.

The malic enzyme (ME) gene is a target for both thyroid hormone receptors and peroxisome proliferator-activated receptors (PPAR). Within the ME promoter, two direct repeat (DR)-1-like elements, MEp and MEd, have been identified as putative PPAR response elements (PPRE). We demonstrate that only MEp and not MEd is able to bind PPAR/retinoid X receptor (RXR) heterodimers and mediate peroxisome proliferator signaling. Taking advantage of the close sequence resemblance of MEp and MEd, we have identified crucial determinants of a PPRE. Using reciprocal mutation analyses of these two elements, we show the preference for adenine as the spacing nucleotide between the two half-sites of the PPRE and demonstrate the importance of the two first bases flanking the core DR1 in 5'. This latter feature of the PPRE lead us to consider the polarity of the PPAR/RXR heterodimer bound to its cognate element. We demonstrate that, in contrast to the polarity of RXR/TR and RXR/RAR bound to DR4 and DR5 elements respectively, PPAR binds to the 5' extended half-site of the response element, while RXR occupies the 3' half-site. Consistent with this polarity is our finding that formation and binding of the PPAR/RXR heterodimer requires an intact hinge T region in RXR while its integrity is not required for binding of the RXR/TR heterodimer to a DR4.

Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.

Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational-experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo.

MMPBSA decomposition of the binding energy throughout a molecular dynamics simulation of Amyloid-Beta (Aß10-35) aggregation

Recent experiments with amyloid-beta (Aß) peptides indicate that the formation of toxic oligomers may be an important contribution to the onset of Alzheimer's disease. The toxicity of Aß oligomers depend on their structure, which is governed by assembly dynamics. However, a detailed knowledge of the structure of at the atomic level has not been achieved yet due to limitations of current experimental techniques. In this study, replica exchange molecular dynamics simulations are used to identify the expected diversity of dimer conformations of Aß10-35 monomers. The most representative dimer conformation has been used to track the dimer formation process between both monomers. The process has been characterized by means of the evolution of the decomposition of the binding free energy, which provides an energetic profile of the interaction. Dimers undergo a process of reorganization driven basically by inter-chain hydrophobic and hydrophilic interactions and also solvation/desolvation processes.

Catechol-binding serines of beta(2)-adrenergic receptors control the equilibrium between active and inactive receptor states.

The binding free energy for the interaction between serines 204 and 207 of the fifth transmembrane helix of the beta(2)-adrenergic receptor (beta(2)-AR) and catecholic hydroxyl (OH) groups of adrenergic agonists was analyzed using double mutant cycles. Binding affinities for catecholic and noncatecholic agonists were measured in wild-type and mutant receptors, carrying alanine replacement of the two serines (S204A, S207A beta(2)-AR), a constitutive activating mutation, or both. The free energy coupling between the losses of binding energy attributable to OH deletion from the ligand and from the receptor indicates a strong interaction (nonadditivity) as expected for a direct binding between the two sets of groups. However, we also measured a significant interaction between the deletion of OH groups from the receptor and the constitutive activating mutation. This suggests that a fraction of the decrease in agonist affinity caused by serine mutagenesis may involve a shift in the conformational equilibrium of the receptor toward the inactive state. Direct measurements using a transient transfection assay confirm this prediction. The constitutive activity of the (S204A, S207A) beta(2)-AR mutant is 50 to 60% lower than that of the wild-type beta(2)-AR. We conclude that S204 and S207 do not only provide a docking site for the agonist, but also control the equilibrium of the receptor between active (R*) and inactive (R) forms.

T-cell hybridoma specific for a cytochrome c peptide: specific antigen binding and interleukin 2 production.

T-cell hybridomas were obtained after fusion of BW 5147 thymoma and long-term cultured T cells specific for cytochrome c peptide 66-80 derivatized with a 2,4-dinitroaminophenyl (DNAP) group. The resulting hybridomas were selected for their capacity to specifically bind to soluble radiolabeled peptide antigen. One T-cell hybrid was positive for antigen binding. This hybrid T cell exhibits surface phenotypic markers of the parent antigen-specific T cells. The binding could be inhibited either by an excess of unlabeled homologous antigen or by cytochrome c peptide 11-25 derivatized with a 2-nitrophenylsulfenyl group. Several other peptide antigens tested failed to inhibit binding of the radioactive peptide. This suggests that a specific amino acid sequence, modified by a DNAP group, is the antigenic structure recognized by the putative T-cell receptor. In addition, direct interaction of DNAP-66-80 peptide with the hybridoma cell line induced production of the T-cell growth factor interleukin 2. Furthermore, supernatants derived from syngeneic macrophages pulsed with the relevant peptide also induced the antigen-specific hybridoma to produce interleukin 2.

Dust Control Using an Asphalt Emulsion, HR-196, 1977

In an effort to control fugitive dust on a gravel surfaced roadway in Boone County, a cationic asphalt emulsion was blended with warm water and applied with an asphalt distributor. The test included various application procedures. After visual observations, it was concluded that this procedure utilizing a dilute asphalt emulsion was not an effective method of dust control.

Development of an Economic Dust Palliative for Limestone Surfaced Secondary Roads, HR-297, 1990

This research project was directed at laboratory and field evaluation of sodium montmorillonite clay (bentonite) as a dust palliative for limestone surfaced secondary roads. It had been postulated that the electrically charged surfaces of the clay particles could interact with the charged surfaces of the limestone and act as a bonding agent to agglomerate fine (-#200) particulates and also to band the fine particulates to larger (+#200) limestone particles. Laboratory testing using soda ash dispersed bentonite treatment of limestone fines indicated significant improvement of compressive strength and slaking characteristics. It was recommended that the project proceed to field trials and test roads were constructed in Dallas and Adair counties in Iowa. Soda ash dispersed bentonite solutions can be field mixed and applied with conventional spray distribution equipment. A maximum of 1.5% bentonite(by weight of aggregate)can be applied at one time. Higher applications would have to be staged allowing the excess moisture to evaporate between applications. Construction of higher application treatments can be accomplished by adding dry bentonite to the surfacing material and then by dry road mixing. The soda ash water solution can then be spray applied and the treated surfacing material wet mixed by motor graders to a consistency of 3 to 4 inch slump concrete. Two motor graders working in tandem can provide rapid mixing for both methods of construction. Calcium and magnesium chloride treatments are 2 to 3 times more effective in dust reduction in the short term (3-4 months) but are prone to washboarding and potholing due to maintenance restrictions. Bentonite treatment at the 2-3% level is estimated to provide a 30-40% dust reduction over the long term(18-24 months). Normal maintenance blading operations can be used on bentonite treated areas. Vehicle braking characteristics are not adversely affected up to the 3.0% treatment level. The bentonite appears to be functioning as a banding agent to bind small particulates to larger particles and is acting to agglomerate fine particles of limestone. This bonding capability appears recoverable from environmental effects of winter, and from alternating wet and dry periods. The bentonite appears to be able to interact with new applications of limestone maintenance material and maintains a dust reduction capability. Soda ash dispersed bentonite treatment is approximately 10 times more cost effective per percent dust reduction than conventional chloride treatments with respect to time. However,the disadvantage is that there is not the initial dramatic reduction in dust generation as with the chloride treatment. Although dust is reduced 30-40% after treatment there is still dust being generated and the traveling public or residents may not perceive the reduction.

Bentonite Treatment for Economical Dust Reduction on Limestone Surfaced Secondary Roads, HR-351, 1995

This research project was directed at laboratory and field evaluation of sodium montmorillonite clay (Bentonite) as a dust palliative for limestone surfaced secondary roads. It was postulated that the electrically charged surfaces (negative) of the clay particles could interact with the charged surfaces (positive) of the limestone and act as a bonding agent to agglomerate fine (-#200) particulates, and also to bond the fine particulates to larger (+#200) limestone particles. One mile test roads were constructed in Tama, Appanoose, and Hancock counties in Iowa using Bentonite treatment levels (by weight of aggregate) ranging from 3.0 to 12.0%. Construction was accomplished by adding dry Bentonite to the surfacing material and then dry road mixing. The soda ash/water solution (dispersing agent) was spray applied and the treated surfacing material wet mixed by motor graders to a consistency of 2 to 3 inch slump concrete. Two motor graders working in tandem provided rapid mixing. Following wet mixing the material was surface spread and compacted by local traffic. Quantitative and qualitative periodic evaluations and testing of the test roads was conducted with respect to dust generation, crust development, roughness, and braking characteristics. As the Bentonite treatment level increased dust generation decreased. From a cost/benefit standpoint, an optimum level of treatment is about 8% (by weight of aggregate). For roads with light traffic, one application at this treatment level resulted in a 60-70% average dust reduction in the first season, 40-50% in the second season, and 20-30% in the third season. Crust development was rated at two times better than untreated control sections. No discernible trend was evident with respect to roughness. There was no evident difference in any of the test sections with respect to braking distance and braking handling characteristics, under wet surface conditions compared to the control sections. Chloride treatments are more effective in dust reduction in the short term (3-4 months). Bentonite treatment is capable of dust reduction over the long term (2-3 seasons). Normal maintenance blading operations can be used on Bentonite treated areas. Soda ash dispersed Bentonite treatment is estimated to be more than twice as cost effective per percent dust reduction than conventional chloride treatments, with respect to time. However, the disadvantage is that there is not the initial dramatic reduction in dust generation as with the chloride treatment. Although dust is reduced significantly after treatment there is still dust being generated. Video evidence indicates that the dust cloud in the Bentonite treated sections does not rise as high, or spread as wide as the cloud in the untreated section. It also settles faster than the cloud in the untreated section. This is considered important for driving safety of following traffic, and for nuisance dust invasion of residences and residential areas. The Bentonite appears to be functioning as a bonding agent.

How do the different components of episodic memory develop? Role of executive functions and short-term feature-binding abilities.

This study investigated the development of all 3 components of episodic memory (EM), as defined by Tulving, namely, core factual content, spatial context, and temporal context. To this end, a novel, ecologically valid test was administered to 109 participants aged 4-16 years. Results showed that each EM component develops at a different rate. Ability to memorize factual content emerges early, whereas context retrieval abilities continue to improve until adolescence, due to persistent encoding difficulties (isolated by comparing results on free recall and recognition tasks). Exploration of links with other cognitive functions revealed that short-term feature-binding abilities contribute to all EM components, and executive functions to temporal and spatial context, although ability to memorize temporal context is predicted mainly by age.

Antigen binding to secretory immunoglobulin A results in decreased sensitivity to intestinal proteases and increased binding to cellular Fc receptors.

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA x antigen immune complexes are selectively transported across Peyer's patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (Fc alphaRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.

Surface Improvement and Dust Palliation of Unpaved Secondary Roads and Streets, HR-151, 1973

As of December 31, 1970 there were 57,270 miles of Local Secondary roads and 32,958 miles of Farm to Market roads in the Iowa secondary road system. The Local Secondary system carried a traffic load of 2,714,180 daily vehicle miles, accounting for 32% of all traffic in the secondary system. For all Local Secondary roads having some form of surfacing, 98% were surfaced with gravel or crushed stone. During the 1970 construction year 335 miles of surfaced roads were constructed in the Local Secondary system with 78% being surfaced with gravel or crushed stone. The total maintenance expenditure for all secondary roads in Iowa during 1970 amounted to $40,086,091. Of this, 42%, or $17,020,332, was spent for aggregate replacement on existing gravel or crushed stone roads with an additional 31% ($12,604,456) being spent on maintenance other than resurfacing. This amounts to 73% of the total maintenance budget and are the largest two maintenance expenditure items out of a list of 10 ranging from bridges to drainage assessments. The next largest item was 7%, for maintenance of existing flexible bases. Three concurrent phases of study were included in this project: (1) laboratory screenings studies of various additives thought to have potential for long-lasting dust palliation, soil additive strength, durability, and additive retention potential; (2) test road construction using those additives that indicated promise for performance-serviceability usage; and (3) observations and tests of constructed sections for evaluation of the additive's contribution to performance and serviceability as well as the relationship to initial costs.

Surface Improvement and Dust Palliation of Unpaved Secondary Roads and Streets, Progress Report, HR-151, 1971

A combined study of dust control and low-cost surface improvements of soil and aggregate materials for immediate (and intermediate) use as a treated surface course is being conducted in three concurrent phases: (1) laboratory screening of various additives thought to have potential for long-lasting dust palliation, soil-additive strength, durability, and additive retention potential; (2) test road construction, using those additives from the screening studies that indicate promise for performance and serviceability; and (3) observation and tests of constructed sections for evaluation of the additive's contribution to performance and serviceability as well as relationship to initial costs. A brief review is presented of the problem, some methods of measuring it, previously adopted approaches to it, project field tests and a portion of the results thus far, and portions of the laboratory work accomplished in the screening studies.

The glucose transporter 4 FQQI motif is necessary for Akt Substrate of 160-Kilodalton-dependent plasma membrane translocation but not Golgi-localized g-ear-containing Arf-binding protein-dependent entry into the insulin-responsive storage compartment.

Newly synthesized glucose transporter 4 (GLUT4) enters into the insulin-responsive storage compartment in a process that is Golgi-localized γ-ear-containing Arf-binding protein (GGA) dependent, whereas insulin-stimulated translocation is regulated by Akt substrate of 160 kDa (AS160). In the present study, using a variety of GLUT4/GLUT1 chimeras, we have analyzed the specific motifs of GLUT4 that are important for GGA and AS160 regulation of GLUT4 trafficking. Substitution of the amino terminus and the large intracellular loop of GLUT4 into GLUT1 (chimera 1-441) fully recapitulated the basal state retention, insulin-stimulated translocation, and GGA and AS160 sensitivity of wild-type GLUT4 (GLUT4-WT). GLUT4 point mutation (GLUT4-F5A) resulted in loss of GLUT4 intracellular retention in the basal state when coexpressed with both wild-type GGA and AS160. Nevertheless, similar to GLUT4-WT, the insulin-stimulated plasma membrane localization of GLUT4-F5A was significantly inhibited by coexpression of dominant-interfering GGA. In addition, coexpression with a dominant-interfering AS160 (AS160-4P) abolished insulin-stimulated GLUT4-WT but not GLUT4-F5A translocation. GLUT4 endocytosis and intracellular sequestration also required both the amino terminus and large cytoplasmic loop of GLUT4. Furthermore, both the FQQI and the SLL motifs participate in the initial endocytosis from the plasma membrane; however, once internalized, unlike the FQQI motif, the SLL motif is not responsible for intracellular recycling of GLUT4 back to the specialized compartment. Together, we have demonstrated that the FQQI motif within the amino terminus of GLUT4 is essential for GLUT4 endocytosis and AS160-dependent intracellular retention but not for the GGA-dependent sorting of GLUT4 into the insulin-responsive storage compartment.