Analysis of Agrobacterium tumefaciens VirB6 and VirB9 of the VirB /D4 type IV secretion system

Agrobacterium tumefaciens uses the VirB/D4 type IV secretion system (T4SS) to translocate oncogenic DNA (T-DNA) and protein substrates to plant cells. Independent of VirD4, the eleven VirB proteins are also essential for elaboration of a conjugative pilus termed the T pilus. The focus of this thesis is the characterization and analysis of two VirB proteins, VirB6 and VirB9, with respect to substrate translocation and T pilus biogenesis. Observed stabilizing effects of VirB6 on other VirB subunits and results of protein-protein interaction studies suggest that VirB6 mediates assembly of the secretion machine and T pilus through interactions with VirB7 and VirB9. Topology studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, a large internal periplasmic loop, five transmembrane segments, and a cytoplasmic C terminus. Topology studies and Transfer DNA immunoprecipitation (TrIP) assays identified several important VirB6 functional domains: (i) the large internal periplasmic loop mediates interaction of VirB6 with the T-DNA, (ii) the membrane spanning region carboxyl-terminal to the large periplasmic loop mediates substrate transfer from VirB6 to VirB8, and (iii) the terminal regions of VirB6 are required for substrate transfer to VirB2 and VirB9. To analyze structure-function relationships of VirB9, the phenotypic consequences of dipeptide insertion mutations were characterized. Substrate discriminating mutations were shown to selectively export the oncogenic T-DNA and VirE2 to plant cells or a mobilizable IncQ plasmid to bacterial cells. Mutations affecting VirB9 interactions with VirB7 and VirB10 were localized to the C- and N- terminal regions respectively. Additionally, “uncoupling” mutations identified in VirB11 and VirB6 that block T pilus assembly, but not substrate transfer to recipient cells, were also identified in VirB9. These results in conjunction with computer analysis establish that VirB9, like VirB6, is also composed of distinct regions or domains that contribute in various ways to secretion channel activity and T pilus assembly. Lastly, in vivo immunofluorescent studies suggest that VirB9 localizes to the outer membrane and may play a role similar to that of secretion/ushers of types II and III secretion systems to facilitate substrate translocation across this final bacterial barrier. ^

Proceedings of the 23rd Annual Biochemical Engineering Symposium

The 23rd Annual Biochemical Engineering Symposium was held at the University of Oklahoma on April 17, 1993. The objectives of the symposium were to provide 1) a forum for informal discussion of biochemical engineering research being carried at the participating universities and 2) an opportunity for students to present and publish their work. Thirteen papers presented at the symposium are included in the proceedings. Because final publication usually takes place in refereed journals, the articles included here are typically brief and often cover work in progress. The program of the symposium and a list of participants are included in the proceedings. ContentsA Low-Cost Bioreactor Strategy for RNA Synthesis, H. Anthony Marble, Eleni Chrisikos, and Robert H. Davis Development of a CELSS Bioreactor: Oxygen Transfer and Micromixing in Parabolic Flight, P.E. Villeneuve, K.S. Wenger, B.G. Thompson, T. Kedar, and E.H. Dunlop Scale-up of Dexter Murine Bone Marrow Cultures Utilizing a Three-Dimensional Fiberglass Support Matrix, John G. Highfill, Paul Todd, Steve Haley, and Dhinaker Kompala Modeling and Estimation of States of Recombinant Fermentations Using Nonlinear Input/Output Models, Vicotr M. Saucedo and M. Nazmul Karim Deadent Microfiltration of Bovine Serum Albumin Suspension Through Yeast Cake Layers and Assymetric Polymeric Membranes, Naveen Arora and Robert H. Davis Monitoring the Fate of Toluene and Phenol in the Rhizosphere, N. Muralidharan, Lawrence C. Davis, and Larry E. Erickson Hydrodynamic Motions Associated with Bubble Coalescence and Breakup, T.Y. Yiin, L.A. Glasgow, and L.E. Erickson Expression and Purification of a-Human Atrial Natriuretic Peptide in Escherichia coli by Fusion with L-Asparaginase, Nien-Tung Ma and Roger G. Harrison High Pressure Crystallization of Proteins, Mungara V. Saikumar, Charles E. Glatz, and Maurice A. Larson Structure/Function Relationships in the Catalytic and Starch Binding Domains of Glucoamylase, Pedro M. Coutinho, Clark Ford, Peter J. Reilly Cellular Responses of Insect Cell Spodoptera frugiperda to Environmental Stresses, Paul Yeh, Grace Y. Sun, Gary A. Weisman, Rakesh Bajpai A Novel Approach to Understanding the Antimicrobial Activity of Peptides, Naveen Pathak, Marie-Helene Janna, Gael Ruche, David McCarthy, and Roger Harrison Mass Transfer in the Bioremediation of Soils Contaminated with Trapped Non-Aqueous Phase Liquids, Xiaoqing Yang, Larry E. Jacobson, and L.T. Fan

Modelización de los Procesos de Cristalización en Poliolefinas

“Por lo tanto, la cristalización de polímeros se supone, y en las teorías se describe a menudo, como un proceso de múltiples pasos con muchos aspectos físico-químicos y estructurales influyendo en él. Debido a la propia estructura de la cadena, es fácil entender que un proceso que es termodinámicamente forzado a aumentar su ordenamiento local, se vea obstaculizado geométricamente y, por tanto, no puede conducirse a un estado de equilibrio final. Como resultado, se forman habitualmente estructuras de no equilibrio con diferentes características dependiendo de la temperatura, presión, cizallamiento y otros parámetros físico-químicos del sistema”. Estas palabras, pronunciadas recientemente por el profesor Bernhard Wunderlich, uno de los mas relevantes fisico-quimicos que han abordado en las ultimas décadas el estudio del estado físico de las macromoléculas, adelantan lo que de alguna manera se explicita en esta memoria y constituyen el “leitmotiv” de este trabajo de tesis. El mecanismo de la cristalización de polímeros esta aun bajo debate en la comunidad de la física de polímeros y la mayoría de los abordajes experimentales se explican a través de la teoría LH. Esta teoría clásica debida a Lauritzen y Hoffman (LH), y que es una generalización de la teoría de cristalización de una molécula pequeña desde la fase de vapor, describe satisfactoriamente muchas observaciones experimentales aunque esta lejos de explicar el complejo fenómeno de la cristalización de polímeros. De hecho, la formulación original de esta teoría en el National Bureau of Standards, a comienzos de la década de los 70, sufrió varias reformulaciones importantes a lo largo de la década de los 80, buscando su adaptación a los hallazgos experimentales. Así nació el régimen III de cristalización que posibilita la creacion de nichos moleculares en la superficie y que dio pie al paradigma ofrecido por Sadler y col., para justificar los experimentos que se obtenian por “scattering” de neutrones y otras técnicas como la técnica de “droplets” o enfriamiento rapido. Por encima de todo, el gran éxito de la teoría radica en que explica la dependencia inversa entre el tamaño del plegado molecular y el subenfriamiento, definido este ultimo como el intervalo de temperatura que media entre la temperatura de equilibrio y la temperatura de cristalización. El problema concreto que aborda esta tesis es el estudio de los procesos de ordenamiento de poliolefinas con distinto grado de ramificacion mediante simulaciones numéricas. Los copolimeros estudiados en esta tesis se consideran materiales modelo de gran homogeneidad molecular desde el punto de vista de la distribución de tamaños y de ramificaciones en la cadena polimérica. Se eligieron estas poliolefinas debido al gran interes experimental en conocer el cambio en las propiedades fisicas de los materiales dependiendo del tipo y cantidad de comonomero utilizado. Además, son modelos sobre los que existen una ingente cantidad de información experimental, que es algo que preocupa siempre al crear una realidad virtual como es la simulación. La experiencia en el grupo Biophym es que los resultados de simulación deben de tener siempre un correlato mas o menos próximo experimental y ese argumento se maneja a lo largo de esta memoria. Empíricamente, se conoce muy bien que las propiedades físicas de las poliolefinas, en suma dependen del tipo y de la cantidad de ramificaciones que presenta el material polimérico. Sin embargo, tal como se ha explicado no existen modelos teóricos adecuados que expliquen los mecanismos subyacentes de los efectos de las ramas. La memoria de este trabajo es amplia por la complejidad del tema. Se inicia con una extensa introducción sobre los conceptos básicos de una macromolecula que son relevantes para entender el contenido del resto de la memoria. Se definen los conceptos de macromolecula flexible, distribuciones y momentos, y su comportamiento en disolución y fundido con los correspondientes parametros caracteristicos. Se pone especial énfasis en el concepto de “entanglement” o enmaranamiento por considerarse clave a la hora de tratar macromoléculas con una longitud superior a la longitud critica de enmaranamiento. Finaliza esta introducción con una reseña sobre el estado del arte en la simulación de los procesos de cristalización. En un segundo capitulo del trabajo se expone detalladamente la metodología usada en cada grupo de casos. En el primer capitulo de resultados, se discuten los estudios de simulación en disolución diluida para sistemas lineales y ramificados de cadena única. Este caso mas simple depende claramente del potencial de torsión elegido tal como se discute a lo largo del texto. La formación de los núcleos “babys” propuestos por Muthukumar parece que son consecuencia del potencial de torsión, ya que este facilita los estados de torsión mas estables. Así que se propone el análisis de otros potenciales que son igualmente utilizados y los resultados obtenidos sobre la cristalización, discutidos en consecuencia. Seguidamente, en un segundo capitulo de resultados se estudian moleculas de alcanos de cadena larga lineales y ramificados en un fundido por simulaciones atomisticas como un modelo de polietileno. Los resultados atomisticos pese a ser de gran detalle no logran captar en su totalidad los efectos experimentales que se observan en los fundidos subenfriados en su etapa previa al estado ordenado. Por esta razon se discuten en los capítulos 3 y 4 de resultados sistemas de cadenas cortas y largas utilizando dos modelos de grano grueso (CG-PVA y CG-PE). El modelo CG-PE se desarrollo durante la tesis. El uso de modelos de grano grueso garantiza una mayor eficiencia computacional con respecto a los modelos atomísticos y son suficientes para mostrar los fenómenos a la escala relevante para la cristalización. En todos estos estudios mencionados se sigue la evolución de los procesos de ordenamiento y de fusión en simulaciones de relajación isoterma y no isoterma. Como resultado de los modelos de simulación, se han evaluado distintas propiedades fisicas como la longitud de segmento ordenado, la cristalinidad, temperaturas de fusion/cristalizacion, etc., lo que permite una comparación con los resultados experimentales. Se demuestra claramente que los sistemas ramificados retrasan y dificultan el orden de la cadena polimérica y por tanto, las regiones cristalinas ordenadas decrecen al crecer las ramas. Como una conclusión general parece mostrarse una tendencia a la formación de estructuras localmente ordenadas que crecen como bloques para completar el espacio de cristalización que puede alcanzarse a una temperatura y a una escala de tiempo determinada. Finalmente hay que señalar que los efectos observados, estan en concordancia con otros resultados tanto teoricos/simulacion como experimentales discutidos a lo largo de esta memoria. Su resumen se muestra en un capitulo de conclusiones y líneas futuras de investigación que se abren como consecuencia de esta memoria. Hay que mencionar que el ritmo de investigación se ha acentuado notablemente en el ultimo ano de trabajo, en parte debido a las ventajas notables obtenidas por el uso de la metodología de grano grueso que pese a ser muy importante para esta memoria no repercute fácilmente en trabajos publicables. Todo ello justifica que gran parte de los resultados esten en fase de publicación. Abstract “Polymer crystallization is therefore assumed, and in theories often described, to be a multi step process with many influencing aspects. Because of the chain structure, it is easy to understand that a process which is thermodynamically forced to increase local ordering but is geometrically hindered cannot proceed into a final equilibrium state. As a result, nonequilibrium structures with different characteristics are usually formed, which depend on temperature, pressure, shearing and other parameters”. These words, recently written by Professor Bernhard Wunderlich, one of the most prominent researchers in polymer physics, put somehow in value the "leitmotiv "of this thesis. The crystallization mechanism of polymers is still under debate in the physics community and most of the experimental findings are still explained by invoking the LH theory. This classical theory, which was initially formulated by Lauritzen and Hoffman (LH), is indeed a generalization of the crystallization theory for small molecules from the vapor phase. Even though it describes satisfactorily many experimental observations, it is far from explaining the complex phenomenon of polymer crystallization. This theory was firstly devised in the early 70s at the National Bureau of Standards. It was successively reformulated along the 80s to fit the experimental findings. Thus, the crystallization regime III was introduced into the theory in order to explain the results found in neutron scattering, droplet or quenching experiments. This concept defines the roughness of the crystallization surface leading to the paradigm proposed by Sadler et al. The great success of this theory is the ability to explain the inverse dependence of the molecular folding size on the supercooling, the latter defined as the temperature interval between the equilibrium temperature and the crystallization temperature. The main scope of this thesis is the study of ordering processes in polyolefins with different degree of branching by using computer simulations. The copolymers studied along this work are considered materials of high molecular homogeneity, from the point of view of both size and branching distributions of the polymer chain. These polyolefins were selected due to the great interest to understand their structure– property relationships. It is important to note that there is a vast amount of experimental data concerning these materials, which is essential to create a virtual reality as is the simulation. The Biophym research group has a wide experience in the correlation between simulation data and experimental results, being this idea highly alive along this work. Empirically, it is well-known that the physical properties of the polyolefins depend on the type and amount of branches presented in the polymeric material. However, there are not suitable models to explain the underlying mechanisms associated to branching. This report is extensive due to the complexity of the topic under study. It begins with a general introduction to the basics concepts of macromolecular physics. This chapter is relevant to understand the content of the present document. Some concepts are defined along this section, among others the flexibility of macromolecules, size distributions and moments, and the behavior in solution and melt along with their corresponding characteristic parameters. Special emphasis is placed on the concept of "entanglement" which is a key item when dealing with macromolecules having a molecular size greater than the critical entanglement length. The introduction finishes with a review of the state of art on the simulation of crystallization processes. The second chapter of the thesis describes, in detail, the computational methodology used in each study. In the first results section, we discuss the simulation studies in dilute solution for linear and short chain branched single chain models. The simplest case is clearly dependent on the selected torsion potential as it is discussed throughout the text. For example, the formation of baby nuclei proposed by Mutukhumar seems to result from the effects of the torsion potential. Thus, we propose the analysis of other torsion potentials that are also used by other research groups. The results obtained on crystallization processes are accordingly discussed. Then, in a second results section, we study linear and branched long-chain alkane molecules in a melt by atomistic simulations as a polyethylene-like model. In spite of the great detail given by atomistic simulations, they are not able to fully capture the experimental facts observed in supercooled melts, in particular the pre-ordered states. For this reason, we discuss short and long chains systems using two coarse-grained models (CG-PVA and CG-PE) in section 3 and 4 of chapter 2. The CG-PE model was developed during the thesis. The use of coarse-grained models ensures greater computational efficiency with respect to atomistic models and is enough to show the relevant scale phenomena for crystallization. In all the analysis we follow the evolution of the ordering and melting processes by both isothermal and non isothermal simulations. During this thesis we have obtained different physical properties such as stem length, crystallinity, melting/crystallization temperatures, and so on. We show that branches in the chains cause a delay in the crystallization and hinder the ordering of the polymer chain. Therefore, crystalline regions decrease in size as branching increases. As a general conclusion, it seems that there is a tendency in the macromolecular systems to form ordered structures, which can grown locally as blocks, occupying the crystallization space at a given temperature and time scale. Finally it should be noted that the observed effects are consistent with both, other theoretical/simulation and experimental results. The summary is provided in the conclusions chapter along with future research lines that open as result of this report. It should be mentioned that the research work has speeded up markedly in the last year, in part because of the remarkable benefits obtained by the use of coarse-grained methodology that despite being very important for this thesis work, is not easily publishable by itself. All this justify that most of the results are still in the publication phase.

Multiscale-fiber-reinforced thermoplastic composites incorporating carbon nanotubes: A review

This article reviews recent literature on hierarchical thermoplastic-based composites that simultaneously incorporate carbon nanotubes (CNTs) and conventional microscale fibers, and discusses the structure?property relationships of the resulting hybrids. The mixing of multiple and multiscale constituents enables the preparation of materials with new or improved properties due to synergistic effects. By exploiting the outstanding mechanical, thermal and electrical properties of CNTs, a new generation of multifunctional high-performance composites suitable for a wide variety of applications can be developed.

Sequencing of 3-O sulfate containing heparin decasaccharides with a partial antithrombin III binding site

Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is ΔU2SHNS,6SI2SHNS,6SI2SHNS,6SIHNAc,6SGHNS,3S,6S (±DDD4–7). We confirmed our results by using integral glycan sequencing and one-dimensional proton NMR. Furthermore, we show that this approach is flexible and is able to derive sequence information on an oligosaccharide mixture. Thus, this methodology will make possible both the analysis of other unusual sequences in HLGAGs with important biological activity as well as provide the basis for the structural analysis of these pharamacologically important group of heparin/heparan sulfates.

PALI—a database of Phylogeny and ALIgnment of homologous protein structures

PALI (release 1.2) contains three-dimensional (3-D) structure-dependent sequence alignments as well as structure-based phylogenetic trees of homologous protein domains in various families. The data set of homologous protein structures has been derived by consulting the SCOP database (release 1.50) and the data set comprises 604 families of homologous proteins involving 2739 protein domain structures with each family made up of at least two members. Each member in a family has been structurally aligned with every other member in the same family (pairwise alignment) and all the members in the family are also aligned using simultaneous super­position (multiple alignment). The structural alignments are performed largely automatically, with manual interventions especially in the cases of distantly related proteins, using the program STAMP (version 4.2). Every family is also associated with two dendrograms, calculated using PHYLIP (version 3.5), one based on a structural dissimilarity metric defined for every pairwise alignment and the other based on similarity of topologically equivalent residues. These dendrograms enable easy comparison of sequence and structure-based relationships among the members in a family. Structure-based alignments with the details of structural and sequence similarities, superposed coordinate sets and dendrograms can be accessed conveniently using a web interface. The database can be queried for protein pairs with sequence or structural similarities falling within a specified range. Thus PALI forms a useful resource to help in analysing the relationship between sequence and structure variation at a given level of sequence similarity. PALI also contains over 653 ‘orphans’ (single member families). Using the web interface involving PSI_BLAST and PHYLIP it is possible to associate the sequence of a new protein with one of the families in PALI and generate a phylogenetic tree combining the query sequence and proteins of known 3-D structure. The database with the web interfaced search and dendrogram generation tools can be accessed at http://pa uling.mbu.iisc.ernet.in/~pali.

A High-Affinity Ca2+ Pump, ECA1, from the Endoplasmic Reticulum Is Inhibited by Cyclopiazonic Acid but Not by Thapsigargin1

To identify and characterize individual Ca2+ pumps, we have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca2+-ATPase homolog in the yeast (Saccharomyces cerevisiae) mutant K616. The mutant (pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca2+ pump and grows very poorly on Ca2+-depleted medium. Membranes isolated from the mutant showed high H+/Ca2+-antiport but no Ca2+-pump activity. Expression of ECA1 in endomembranes increased mutant growth by 10- to 20-fold in Ca2+-depleted medium. 45Ca2+ pumping into vesicles from ECA1 transformants was detected after the H+/Ca2+-antiport activity was eliminated with bafilomycin A1 and gramicidin D. The pump had a high affinity for Ca2+ (Km = 30 nm) and displayed two affinities for ATP (Km of 20 and 235 μm). Cyclopiazonic acid, a specific blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, inhibited Ca2+ transport (50% inhibition dose = 3 nmol/mg protein), but thapsigargin (3 μm) did not. Transport was insensitive to calmodulin. These results suggest that this endoplasmic reticulum-type Ca2+-ATPase could support cell growth in plants as in yeast by maintaining submicromolar levels of cytosolic Ca2+ and replenishing Ca2+ in endomembrane compartments. This study demonstrates that the yeast K616 mutant provides a powerful expression system to study the structure/function relationships of Ca2+ pumps from eukaryotes.

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety.

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

Negative-strand RNA viruses: genetic engineering and applications.

The negative-strand RNA viruses are a broad group of animal viruses that comprise several important human pathogens, including influenza, measles, mumps, rabies, respiratory syncytial, Ebola, and hantaviruses. The development of new strategies to genetically manipulate the genomes of negative-strand RNA viruses has provided us with new tools to study the structure-function relationships of the viral components and their contributions to the pathogenicity of these viruses. It is also now possible to envision rational approaches--based on genetic engineering techniques--to design live attenuated vaccines against some of these viral agents. In addition, the use of different negative-strand RNA viruses as vectors to efficiently express foreign polypeptides has also become feasible, and these novel vectors have potential applications in disease prevention as well as in gene therapy.

Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.

Macromolecular interactions define many biological phenomena. Although genetic methods are available to identify novel protein-protein and DNA-protein interactions, no genetic system has thus far been described to identify molecules or mutations that dissociate known interactions. Herein, we describe genetic systems that detect such events in the yeast Saccharomyces cerevisiae. We have engineered yeast strains in which the interaction of two proteins expressed in the context of the two-hybrid system or the interaction between a DNA-binding protein and its binding site in the context of the one-hybrid system is deleterious to growth. Under these conditions, dissociation of the interaction provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two-hybrid system" and "reverse one-hybrid system" facilitate the study of the structure-function relationships and regulation of protein-protein and DNA-protein interactions. They should also facilitate the selection of dissociator molecules that could be used as therapeutic agents.

Rational interleukin 2 therapy for HIV positive individuals: daily low doses enhance immune function without toxicity.

When administered in high doses to HIV positive (HIV+) individuals, interleukin 2 (IL-2) causes extreme toxicity and markedly increases plasma HIV levels. Integration of the information from the structure-activity relationships of the IL-2 receptor interaction, the cellular distribution of the different classes of IL-2 receptors, and the pharmacokinetics of IL-2 provides for the rationale that low IL-2 doses should circumvent toxicity. Therefore, to identify a nontoxic, but effective and safe IL-2 treatment regimen that does not stimulate viral replication, doses of IL-2 from 62,500 to 250,000 IU/m2/day were administered subcutaneously for 6 months to 16 HIV+ individuals with 200-500 CD4+ T cells/mm3. IL-2 was already detectable in the plasma of most HIV+ individuals even before therapy. Peak plasma IL-2 levels were near saturating for high affinity IL-2 receptors in 10 individuals who received the maximum nontoxic dose, which ranged from 187,500 to 250,000 IU/m2/day. During the 6 months of treatment at this dose range, plasma levels of proinflammatory cytokines remained undetectable, and plasma HIV RNA levels did not change significantly. However, delayed type hypersensitivity responses to common recall antigens were markedly augmented, and there were IL-2 dose-dependent increases in circulating Natural Killer cells, eosinophils, monocytes, and CD4+ T cells. Expanded clinical trials of low dose IL-2 are now warranted, especially in combination with effective antivirals to test for the prevention of immunodeficiency and the emergence of drug-resistant mutants and for the eradication of residual virions.

Heterodimer formation and activity in the human enzyme galactose-1-phosphate uridylyltransferase.

One of the fundamental questions concerning expression and function of dimeric enzymes involves the impact of naturally occurring mutations on subunit assembly and heterodimer activity. This question is of particular interest for the human enzyme galactose-l-phosphate uridylyl-transferase (GALT), impairment of which results in the inherited metabolic disorder galactosemia, because many if not most patients studied to date are compound heterozygotes rather than true molecular homozygotes. Furthermore, the broad range of phenotypic severity observed in these patients raises the possibility that allelic combination, not just allelic constitution, may play some role in determining outcome. In the work described herein, we have selected two distinct naturally occurring null mutations of GALT, Q188R and R333W, and asked the questions (i) what are the impacts of these mutations on subunit assembly, and (ii) if heterodimers do form, are they active? To answer these questions, we have established a yeast system for the coexpression of epitope-tagged alleles of human GALT and investigated both the extent of specific GALT subunit interactions and the activity of defined heterodimer pools. We have found that both homodimers and heterodimers do form involving each of the mutant subunits tested and that both heterodimer pools retain substantial enzymatic activity. These results are significant not only in terms of their implications for furthering our understanding of galactosemia and GALT holoenzyme structure-function relationships but also because the system described may serve as a model for similar studies of other complexes composed of multiple subunits.

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

A paradigm for drug discovery employing encoded combinatorial libraries.

Very large combinatorial libraries of small molecules on solid supports can now be synthesized and each library element can be identified after synthesis by using chemical tags. These tag-encoded libraries are potentially useful in drug discovery, and, to test this utility directly, we have targeted carbonic anhydrase (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1) as a model. Two libraries consisting of a total of 7870 members were synthesized, and structure-activity relationships based on the structures predicted by the tags were derived. Subsequently, an active representative of each library was resynthesized (2-[N-(4-sulfamoylbenzoyl)-4'-aminocyclohexanespiro]-4-oxo-7 -hydroxy- 2,3-dihydrobenzopyran and [N-(4-sulfamoylbenzoyl)-L-leucyl]piperidine-3-carboxylic acid) and these compounds were shown to have nanomolar dissociation constants (15 and 4 nM, respectively). In addition, a focused sublibrary of 217 sulfamoylbenzamides was synthesized and revealed a clear, testable structure-activity relationship describing isozyme-selective carbonic anhydrase inhibitors.