Tricyclic azepine derivatives as selective brain penetrant 5-HT6 receptor antagonists.

Starting from a benzazepine sulfonamide 5-HT(6) receptor antagonist lead with limited brain penetration, application of a strategy of conformational constraint and reduction of hydrogen bond donor count led to a novel series of tricyclic derivatives with high 5-HT(6) receptor affinity and excellent brain:blood ratios.

Involvement of an altered 5-HT -{6} receptor function in behavioral symptoms of Alzheimer's disease.

We studied the hypothesis that disturbances in 5-HT_{6} receptor function in the temporal cortex may contribute to clinical symptoms of Alzheimer's disease (AD). 5-HT_{6} density and 5-HT levels were significantly decreased in a cohort of AD patients prospectively assessed for cognitive/behavioral symptoms. cAMP formation after stimulation with the selective 5-HT_{6} receptor agonist E-6801 was significantly lower (p<0.01) in AD (170.02 +/- 27.53 pmol/mg prot.) compared to controls (823.33 +/-196.67). In addition, the ratio cAMP formation after stimulation with E-6801/5-HT_{6} receptor density was significantly lower (p< 0.01) in AD (6.67 +/- 0.83) compared to controls (16.67 +/- 3.33). Splitting these results by sex, 5-HT_{6} receptor activation was significantly lower (p< 0.01) in AD females compared to males (121.67 +/- 30.02 vs. 231.67 +/- 34.17 pmol/mg prot). 5-HT_{6} density and 5-HT levels were significantly correlated (p < or = 0.01) in both controls and AD patients, although in AD, this correlation was lost in females. Psychosis factor was the best predictor of reduced 5-HT levels or adenylate cyclase activity after E-6801 stimulation, the former result being due to females. It may be suggested that psychotic symptoms may be related to a dysregulation of 5-HT_{6} activation by 5-HT in the temporal cortex. These results are discussed in terms of purported influence of sex and therapeutical approaches to psychosis in AD.

Characterization of scFv-Ig constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths.

The heavy (VH) and light (VL) chain variable regions of the murine anti-human CD20 mAb 1F5 were cloned, and four single-chain Ab (scFv) molecules were constructed using linker peptides of variable lengths to join the VH and VL domains. Three constructs were engineered using linker peptides of 15, 10, and 5 aa residues consisting of (GGGGS)3, (GGGGS)2, and (GGGGS)1 sequences, respectively, whereas the fourth was prepared by joining the VH and VL domains directly. Each construct was fused to a derivative of human IgG1 (hinge plus CH2 plus CH3) to facilitate purification using staphylococcal protein A. The aggregation and CD20 binding properties of these four 1F5 scFv-Ig derivatives produced were investigated. Both size-exclusion HPLC column analysis and Western blots of proteins subjected to nonreducing SDS-PAGE suggested that all four 1F5 scFv-Ig were monomeric with m.w. of approximately 55 kDa. The CD20 binding properties of the four 1F5 scFv-Ig were studied by ELISA and flow cytometry. The 1F5 scFv-Ig with the 5-aa linker (GS1) demonstrated significantly superior binding to CD20-expressing target cells, compared with the other scFv-Ig constructs. Scatchard analysis of the radiolabeled monovalent GS1 scFv-Ig revealed a binding avidity of 1.35 x 108 M-1 compared with an avidity of 7.56 x 108 M-1 for the native bivalent 1F5 Ab. These findings suggest that the GS1 scFv-Ig with a short linker peptide of approximately 5 aa is the best of the engineered constructs for future studies.

Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes.

The combination of anti-CD2 mAb 9.6 and 9-1, specific for distinct epitopes, induces proliferation of resting human T cells. The mitogenic activity of this mAb mixture depends upon accessory cells and the 9-1 mAb Fc domain. To further study the functional properties of these mAb, their variable regions were cloned and expressed as monospecific single-chain Fv (scFv) proteins fused to the human IgG1 Fc domain (scFvIg). A novel bispecific scFvIg was constructed by cloning the two monospecific scFv binding sites in tandem, with the 9.6 scFv placed N-terminal to the 9-1 scFvIg. Monospecific scFvIg binding to CD2 was comparable to that of the corresponding parental mAb, while the bispecific scFvIg exhibited binding activity similar to that of the 9-1 scFvIg. The combination of 9.6 scFvIg and 9-1 mAb was mitogenic, whereas mixtures including the 9-1 scFvIg were non-stimulatory, confirming the unique properties of the 9-1 IgG3 Fc. Without the IgG3 tail, the bispecific 9.6/9-1 scFvIg was directly mitogenic and was a more potent mitogen than the mAb mixture, but was accessory cell dependent. Unlike the combination of mAb, the bispecific reagent did not directly mobilize calcium in T cells. In comparison to the mAb mixture, bispecific 9.6/9-1 scFvIg-mediated stimulation of a mixed lymphocyte reaction was significantly more resistant to inhibition of the CD28 co-stimulatory pathway by the inhibitor CTLA-4-Ig. These results show that expression of the 9.6 and 9-1 binding sites together on a bispecific scFvIg increased the mitogenic properties of the mAb and altered the degree of accessory cell signals required for T cell activation.

Costimulation by CD28 sFv expressed on the tumor cell surface or as a soluble bispecific molecule targeted to the L6 carcinoma antigen.

Interaction of the CD80 (B7-1) and CD86 (B7-2) molecules on antigen presenting cells with the receptors CD28 and CTLA-4 on T cells generates signals important in the regulation of immune responses. Because this receptor system involves multiple receptor-ligand interactions, determining the function for individual receptors has been difficult. One approach is the use of antibodies and their derivatives with singular specificity as substitute ligands to explore the activities of these molecules. We have constructed recombinant mono- and bi-specific sFv molecules specific for the CD28 receptor that are capable of binding and generating costimulatory signals to activate T cells. We demonstrate that these soluble molecules are capable of higher levels of costimulation than soluble CD80Ig at equivalent concentrations. We also constructed artificial adhesion receptors on the cell surface using two different CD28-specific sFvIgs fused to the CD80 cytoplasmic and transmembrane domains. In this report, we compared costimulation by a soluble bispecific (alpha CD28-alpha L6) single chain sFvIg fusion protein to that generated by L6 antigen positive (L6+) H3347 tumor cells transduced with cell surface expressed forms of alpha CD28 sFv's. We show that the bispecific protein can target potent CD28 costimulatory activity to L6+ tumor cells in vitro. We also show that transfection of the cell surface forms of the two different CD28 sFvIgs into H3347 tumor cells allows them to generate significant costimulatory signals to activated T cells. Finally, we demonstrate that tumor cell presentation of either the soluble bispecific or transduced cell surface sFv generate similar costimulatory effects resulting in T cell activation.

Rapid and reliable cloning of antibody variable regions and generation of recombinant single chain antibody fragments.

Single chain antibody variable region fragments (sFv), by virtue of their size and method of construction are potentially useful as therapeutic reagents and as tools for exploring cell surface receptor function. sFv offer several advantages over the intact immunoglobulin molecule. For instance, they are expressed from a single transcript and can be molecularly linked to other proteins to generate bispecific sFv molecules or single-chain immunotoxins. The relatively small size of sFv is an advantage in allowing for easier penetrance into tissue spaces, and their clearance rate is exceedingly rapid. sFv are useful for gene therapy since they can be directed to a specific cellular localization and can be fused to retroviral env genes to control viral host range. To prepare sFv to murine and human leukocyte CD antigens, we devised a method for rapid cloning and expression that can yield functional protein within 2-3 weeks of RNA isolation from hybridoma cells. The variable regions were cloned by poly-G tailing the first strand cDNA followed by anchor PCR with a forward poly-C anchor primer and a reverse primer specific for constant region sequence. Both primers contain flanking restriction sites for insertion into PUC19. Sets of PCR primers for isolation of murine, hamster and rat VL and VH genes were generated. Following determination of consensus sequences for a specific VL and VH pair, the VL and VH genes were linked by DNA encoding an intervening peptide linker [usually (Gly4Ser)3] and the VL-link-VH gene cassettes were transferred into the pCDM8 mammalian expression vector. The constructs were transfected into COS cells and sFvs were recovered from spent culture supernatant. We have used this method to generate functional sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45 and to murine CD3 and gp39, from hybridomas producing murine, rat, or hamster antibodies. Initially, the sFvs were expressed as fusion proteins with the hinge-CH2-CH3 domains of human IgG1 to facilitate rapid characterization and purification using goat anti-human IgG reagents or protein A. We also found that active sFv could be expressed with a small peptide > or = tag > or = or in a tail-less form. Expression of CD3 (G19-4) sFv tail-less or Ig tailed forms demonstrated increased cellular signalling activity and suggested that sFv have potential for activating receptors.

Research and development of active control systems

Research and development of full-scale active control systems has been a main focus of this research project involving researches and engineers from MTS Systems Corporation of Minneapolis, the Takenaka Corporation and Kayaba Industries of Japan, as well as NCEER researches. The major objective was to develop two full-scale active systems, an active bracing systems and an active mass damper systems, so that implementational issues could be identified and addressed, their perfomance under actual wind loads and earthquakes could be evaluated, and design guidelines for these systems could be developed. Through a carefully planned analytical and experimental program, this research effort has led to implementation of these systems in full-scale structures. Observed perfomance of these systems under actual wind and earthquake loads shows that the concept of active control, originated more than 20 years ago, has led to the successful development of active devices for civil engineering structural control.(AU)

Seismic applications of viscoelastic dampers

The application of viscoelastic materials to vibration control can be dated back to the 1950's, when they were first used on aircrafts as a means of controlling vibration-induced fatigue in airframes. Its application to civil engineering structures appears to have begun in 1969 when 10,000 viscoelastic dampers were installed in each of the twin towers of the World Trade Center in New York to help resist wind loads. The major objective of this research project was to investigate the feasibility of using viscoelastic dampers in structures to protect against seismic loads.(AU)

Seismic protective systems for computers and data processing equipment

Data processing installations, intelligent workstations, personal computers and related types of equipment are vital to the successfull operation of business, education, research, service and industrial operations in today's world. Any interruption in the operation of data processing or computer equipment can halt or significantly impact the operation of business activity. Especially important are such items as on-line banking, process controls, communications, hospitals and emergency facilities. This scenario, coupled with the occurrence of an earthquake, can lead to total chaos if the coputers fail due to the earthquake shock and vibration effects.(AU)

Code development for nonstructural components

The importance of nonstructural component issues in seismic design and perfomance evaluation is now well recognized. Today, major building codes and seismic design guidelines exist which address seismic design forces for various nonstructural components,. In these provisions, the design force is formulated as an equivalent static lateral force applied to the approximate center of gravity of the component being considered.(AU)

An energy approach to seismic analysis and design of secondary systems

The dynamic analysis and desugn of secondary systems have been extensively studied over the last two decades, resulting in a better understanding of their general dynamic characteristics. One of the current challenges for researches is to develop simple yet accurate procedures incorporating these research results and transfer them into the development of practical design and perfomance evaluation procedures. This is the basic thrust of this report. Statistical energy analysis has been proven to be a powerful tool in the dynamic analysis of complex systems involving interaction effect between acoustic field and structure. In this report, such a tool is systematically introduced to simplify the analysis and design procedures of secondary systems. This investigation starts out with the identification od special problems and assumption verification associated with the extension of its application. The relation between power flow trnasmitted from one system to another and energies stored in two systems which are commonly encountered in civil engineering. The concept of dissipate and penetrating power flow is developed to characterize the dissipating and transmitting properties of the coupling element. The relationship developed in a generic system is then applied to a simple primary-secondary system to investigate the general behavior of power flow and energy quantities. Thier equivalence to the conventional response variables such as relative displacement and absolute acceleration is demonstrated analytically as well as through numerical examples. For a general complex system in which many high-frequency modes are excited by the external excitation, a simple procedure in statistical energy anlysis is directly applicable. For intermediate cases commonly encountered in civil engineering where a few low-frequency modes in primary-secondary system are excited by external forces, a mean-square condensation method is developed to condense the number of degrees of freedom step-by-step through energy equivalence before and after condensation. Closed-form formulation used in the condensation process are derived so that response calculations can be expedited. The power flow and energy analyses are further extended to a class of complex primary-secondary systems for which the interaction effect between different branches of the secondary system is thoroughly studied; optimum damping of the secondary system is recognized as in the simple primary system and the dynamic characteristics of multi-tuned primary-secondary systems are investigated. The exact solution for this class of complex systems can also serve to assess many types of approximated schemes proposed in the past. A decoupling criterion for the dynamic response of secondary systems is systematically established. The question about which response characteristics (primary or secondary) are more sensitive to the decoupling action id first raised and studied. The conservative domains in which non-interaction analyses give rise to overestimated results for different systems are investigated and compared under different conditions. Sufficient conditions fro dynamic decoupling of secondary systems are also developed in this report.(AU)

Seismic behavior and design guidelines for steel frame structures with added viscoelastic dampers

In order to determine the effectiveness of adding viscoelastic dampers to structure on the reduction of their seismic response, a comprehensive analytical experimental program was carried out. The experimental program was first conducted on a 2/5-scale five-story steel frame under precisely controlled ambient temperatures and subject to simulated ground motions with peak accelerations ranging from 0.12g to 0.60g. Results show that viscoelastic dampers are very effective in attenuating seismic structural response at all levels of earthquake ground motions, and that their energy dissipation capacity decreases as ambient temperature increases. However, they are effective at all temperatures tested in the research program. A rational seismic design procedure for viscoelastically damped structure is developed based on these results. Further tests using a full-scale prototype structure confirm that damping in the full-scale structure can be significantly increased by adding relatively small viscoelastic dampers. The damper design procedure developed based on the scaled model can also be applied to the full-scale structure. This full-scale analytical and experimental study provides an important base for applying the extensive data generated from the scaled model testing to the full-scale structures.(AU)

Seismic testing of intallation methods for computers and data processing equipment

The basic objective of the test program described in this report was to investigate seismic response of computers and data processing equipment and to evaluate a variety of possible earthquake resistant installation methods ranging from those currently in use to more advanced designs using energy absorbing materials. This report documents results and assesses their significant based on three two-week test sessions performed jointly by NCEER and IBM investigators from June, 1991 through June, 1992. These tests were conducted on different mainframe systems utilizing a variety of earthquake inputs and different installation methods. The equipment response to the earthquake inputs is analyzed to enable a quatitative evaluation of the installation methods. Discussions and conclusions regarding the equipment sensitivity to earthquakes and the performance of selected installation methods are presented (AU)

Inelastic response of reinforced concrete structures with viscoelastic braces

The adition of viscoelastic braces in structures for vibration reduction was proposed and implemented in the past decade in metal models or full-scale structures. Viscoelastic braces provide energy dissipation, while the structures remains by-and-large elastic. In reinforced concrete structures, the seismic response in by-and-large inelastic, which is often accompanied by permanent deformations and damage. The addition of viscoelastic dampers can dissipate energy at the early stages of cracking of the concrete elemnts and reduce the development of damage. With proper selectio of dampers, this damage can be substantially reduced or even eliminated. However the addition of viscoelastic dampers may stiffen the structure unnecessarily producing increased inertial forces and base shears when subjected to seismic motion. The quantification of the influence of viscous and elastic stiffness properties of dampers during the inelastic response of reinforced concrete structures is the subject of this invetigation. Models for analysis of inelastic response with damege indexing for reinforced concrete structures that include viscoelastic braces are developed and calibrated using experimental data produced by shaking table tests. These models are then used to determine the variation of expected damage in the presence of damping and quantify the hysteretic energy dissipation along with the damping energy (AU)

Assessment of the 1991 NEHRP provisions for nonstructural components and recommended revisions

As one part of the 1994 update effort of the 1991 NEHRP provisions, the seismic design force formulas for nonstructural components as they exist in the 1991 provisions are critically assessed and some of their shortcommings are identified. Various levels of improvements to these formulas are then presented which, on the one hand, preserve the equivalent lateral force format for design applicability and, on the other, correct some of their deficiencies on the basis of analyses, experimental results and observation data from past earthquakes. Based on different interpretations of the component seismic coefficients as well as different degrees of simplicity required in practical design, three recommendations are proposed. The first recommended revision is the most comprehensive in that both effects of nonstructural component anchorage detailing and its supporting structural characteristics are taken into account. The second recommendation is a structure-driven type of modification of the current provisions and is motivated by the possibility that nonstructural component information during a design process is not available. The third revision, however, mainly concentrates on the effect of nonstructural component characteristics on the design force although it partially implies structural effects in the process of determining the response modification coefficient. The maximum and minimum design forces in the three recommendations are compared with those produced by the 1991 NEHRP provisions, the 1991 UBC, and the 1985 Tri-Service codes. Case studies of a parapet, a storage rack and a general equipment attached to a reinforced concrete shear wall structure are provided to show the relative conservatism involved in different codes and the importance of the factors ignored in the current provisions. Simple displacement equations are also developed in this report to provide deformation information needed in some cases of practical design (AU)

Tyrosine phosphorylation of CD6 by stimulation of CD3: augmentation by the CD4 and CD2 coreceptors.

When T cells are activated via the T cell receptor (TCR) complex a number of cellular substrates, including some cell surface proteins, become phosphorylated on tyrosine (Tyr) residues. Phosphorylation of cytoplasmic Tyr renders these cell surface receptors competent to interact with proteins that link cell surface receptors to protein in the intracellular signaling pathways. Here we show that Tyr residues in the cytoplasmic domain of CD6 become phosphorylated upon T cell activation via the TCR complex. Tyr phosphorylation was observed when the T cells were activated by crosslinking CD3 or by cocrosslinking CD3 with CD2 or CD4, but not when the cells were stimulated by crosslinking CD2, CD4, or CD28 alone. Unlike other Tyr kinase substrates, such as the phospholipase C gamma 1-associated pp35/36 protein, whose level of Tyr phosphorylation is highest when T cells are activated by cocrosslinking CD3 with CD2, the levels of CD6 Tyr phosphorylation are highest when T cells were activated by cocrosslinking CD3 with CD4.

Active bracing system : A full scale implementation of active control

An active bracing system has been designed, fabricated, and installed in a full-scale dedicated test structure for structural response control under seismic loads. This report presents (i) a description of the constructed system, (ii) design specifications for the control system along with simulation studies for the design earthquake, and (iii) observed performance of the system under three actual earthquakes and other artificial loadings. Detailed design and analysis of the active system are carried out with respect to hardware development, control force constraints, and power and energy requirements. It is shown that a full-scale efficient active structural control system can be developed within limits of current technology. Simulation results provide information on performace bounds that can be expected of active systems in structural control under seismic loads and under constraints imposed by practical considerations. Installation details of the system in the building structure are presented along with the selections for fail-safe shutdown operations in case of malfunctions. Also presented are the procedures for proper maintenance and self testing which ensure continuous control with minimal resources. The observed performance under artificial loadings and actual ground motions is compared with the estimated analytical response. It is shown that the performance of the active bracing system is predictable by simple analytical procedures and efficient within the design limitations(AU)

Active control for bridge applications : Case studies

Active control research for structures against large environmental loads has received considerable attention in recent years. Large-scale laboratory experimentation and the development of full-scale active protective systems have taken place in the area of building structures. For bridge applications, however, the feasibility of using active control has been limited to studies of analytical and simulation nature. In this paper, three case studies of active bridge control are summarized in order to provide added impetus to conducting more in-depth studies of active control as a possible protective systems principle for bridges against earthquakes and other environmental loads.(AU)