Mutagenic dissection of hemoglobin cooperativity: effects of amino acid alteration on subunit assembly of oxy and deoxy tetramers.

Free energies of oxygen-linked subunit assembly and cooperative interaction have been determined for 34 molecular species of human hemoglobin, which differ by amino acid alterations as a result of mutation or chemical modification at specific sites. These studies required the development of extensions to our earlier methodology. In combination with previous results they comprise a data base of 60 hemoglobin species, characterized under the same conditions. The data base was analyzed in terms of the five following issues. (1) Range and sensitivity to site modifications. Deoxy tetramers showed greater average energetic response to structural modifications than the oxy species, but the ranges are similar for the two ligation forms. (2) Structural localization of cooperative free energy. Difference free energies of dimer-tetramer assembly (oxy minus deoxy) yielded delta Gc for each hemoglobin, i.e., the free energy used for modulation of oxygen affinity over all four binding steps. A structure-energy map constructed from these results shows that the alpha 1 beta 2 interface is a unique structural location of the noncovalent bonding interactions that are energetically coupled to cooperativity. (3) Relationship of cooperativity to intrinsic binding. Oxygen binding energetics for dissociated dimers of mutants strongly indicates that cooperativity and intrinsic binding are completely decoupled by tetramer to dimer dissociation. (4) Additivity, site-site coupling and adventitious perturbations. All these are exhibited by individual-site modifications of this study. Large nonadditivity may be correlated with global (quaternary) structure change. (5) Residue position vs. chemical nature. Functional response is solely dictated by structural location for a subset of the sites, but varies with side-chain type at other sites. The current data base provides a unique framework for further analyses and modeling of fundamental issues in the structural chemistry of proteins and allosteric mechanisms.


Regulation of oxygen affinity by quaternary enhancement: does hemoglobin Ypsilanti represent an allosteric intermediate?

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.


Crystal structure of human immunoglobulin fragment Fab New refined at 2.0 A resolution.

The three-dimensional structure of the human immunoglobulin fragment Fab New (IgG1, lambda) has been refined to a crystallographic R-factor of 16.9% to 2 A resolution. Rms deviations of the final model from ideal geometry are 0.014 A for bond distances and 3.03 degrees for bond angles. Refinement was based on a new X-ray data set including 28,301 reflections with F > 2.5 sigma(F) from 6.0 to 2.0 A resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide-chain folding in the third complementarity-determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CH1. Amino acid sequence changes were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results in an improved VH framework model for the "humanization" of monoclonal antibodies.


Limits on alpha-helix prediction with neural network models.

Using a backpropagation neural network model we have found a limit for secondary structure prediction from local sequence. By including only sequences from whole alpha-helix and non-alpha-helix structures in our training and test sets--sequences spanning boundaries between these two structures were excluded--it was possible to investigate directly the relationship between sequence and structure for alpha-helix. A group of non-alpha-helix sequences, that was disrupting overall prediction success, was indistinguishable to the network from alpha-helix sequences. These sequences were found to occur at regions adjacent to the termini of alpha-helices with statistical significance, suggesting that potentially longer alpha-helices are disrupted by global constraints. Some of these regions spanned more than 20 residues. On these whole structure sequences, 10 residues in length, a comparatively high prediction success of 78% with a correlation coefficient of 0.52 was achieved. In addition, the structure of the input space, the distribution of beta-sheet in this space, and the effect of segment length were also investigated.


Comparative molecular modeling and crystallization of P-30 protein: a novel antitumor protein of Rana pipiens oocytes and early embryos.

The P-30 protein (Onconase) of Rana pipiens oocytes and early embryos is homologous to members of the pancreatic ribonuclease superfamily and exhibits an antitumor activity in vitro and in vivo. It appears that the ribonucleolytic activity of P-30 protein may be required for its antitumor effects. A comparative molecular model of P-30 protein has been constructed based upon the known three-dimensional structure of bovine pancreatic RNase A in order to provide structural information. Functionally, these enzymes hydrolyze oligoribonucleotides to pyrimidine-3'-phosphate monoesters and 5'-OH ribonucleotides. In the modeling procedure, automated sequence alignments were revised based upon the inspection of the RNase A structure before the amino acids of the P-30 protein were assigned the coordinates of the RNase A template. The inevitable intermolecular steric clashes that result were relieved on an interactive graphics device through the adjustment of side chain torsion angles. This process was followed by energy minimization of the model, which served to optimize stereochemical geometry and to relieve any remaining unacceptably close contacts. The resulting model retains the essential features of RNase A as sequence insertions and deletions are almost exclusively found in exposed surface loops. The all atom superposition of active site residues of the P-30 protein model and an identically minimized RNase A structure has a root mean square deviation of 0.52 A. Though tentative, the model is consistent with a pyrimidine specificity.(ABSTRACT TRUNCATED AT 250 WORDS)


Structure of a rhombohedral R6 insulin/phenol complex.

Hexameric insulin has been crystallized from different conditions in a variety of crystalline modifications. In the presence of approximately 1% phenol and at a pH of 8.5, a new rhombohedral form is produced, space group R3, a = 79.92A and c = 40.39A, in which the asymmetric unit consists of a dimer. The structure has been solved and refined, using data between 8.0 and 2.5A resolution, to a residual of 0.157. The two monomers in the asymmetric unit have nearly identical R conformations, that is, residues B1 through B8 are alpha-helical, producing a continuous alpha-helix from B1 through B19. A phenol molecule is hydrogen bonded to the carbonyl oxygen of A6 Cys of each monomer. Small differences in conformation and the final (2Fo-Fc) and difference electron density maps suggest that an additional phenol molecule is coordinated to one of the two zinc ions.


Folding protein alpha-carbon chains into compact forms by Monte Carlo methods.

A method is presented for generating folded chains of specific amino acid sequences on a simple cubic lattice. Monte Carlo simulations are used to transform extended geometries of simplified alpha-carbon chains for eight small monomeric globular proteins into folded states. Permitted chain transitions are limited to a few types of moves, all restricted to occur on the lattice. Crude residue-residue potentials derived from statistical structure data are used to describe the energies for each conformer. The low resolution structures obtained by this procedure contain many of the correct gross features of the native folded architectures with respect to average residue energy per nonbonded contact, segment density, and location of surface loops and disulfide pairs. Rms deviations between these and the native X-ray structures and percentage of native long-range contacts found in these final folded structures are 7.6 +/- 0.7 A and 48 +/- 3%, respectively. This procedure can be useful for predicting approximate tertiary interactions from amino acid sequence.


What is the pitch of the alpha-helical coiled coil?

The alpha-helical, coiled-coil protein motif is increasingly recognized in a variety of functional classes of proteins. The pitch of a coiled coil, or rate of winding of the alpha-helices around each other, is a key determinant of both intra- and intermolecular interactions. Experimental measurements of the pitch of parallel two-stranded coiled coils of muscle proteins, and examination of the recently determined structure of another two-stranded coiled coil, the GCN4 transcription factor protein, suggest that the pitch has an average value of about 140 A. This value is consistent with the observed number of residues per turn in alpha-helices of globular proteins, the determinant of the interhelical packing within the coiled-coil motif. An understanding of the structural determinants of this value for the pitch and possible variations will be important in defining the interactions of coiled-coil proteins with other macromolecules.


Generation of a substructure library for the description and classification of protein secondary structure. I. Overview of the methods and results.

Protein secondary structure has been typically classified into four major classes--alpha-helices, extended strands, reverse turns, and loops. Available methods for secondary structure analysis utilize predefined structure templates to search for structural matches among proteins. By this approach a significant portion of a proteins backbone conformation is assigned to one of a limited number of conformations or, if unassigned, to random coil. To expand our ability to describe protein secondary structure, we have developed an algorithm that operates independently of a predefined structure template. The procedure uses two geometric descriptors, the linear distance and the backbone dihedral angle, to represent the conformation form the alpha-carbon coordinates. The algorithm functions by searching for conformationally equivalent, contiguous fragments without regard to secondary structural classification and is thus independent of the complexity of the backbone fold. The result is a library of conformationally equivalent structure fragments that exhibit some novel characteristics. The library contains features that reproduce the major secondary structure classes as well as defining conformations previously described only as random or undefined conformations. Additionally, the library defines several subclassifications of beta-strands. We present here a validation of this method and a presentation and discussion of the most significant results. In a second study, we report the results of application of this method to spectra-structure correlations in Fourier transform infrared spectroscopy.


Generation of a substructure library for the description and classification of protein secondary structure. II. Application to spectra-structure correlations in Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy has become well known as a sensitive and informative tool for studying secondary structure in proteins. Present analysis of the conformation-sensitive amide I region in protein infrared spectra, when combined with band narrowing techniques, provides more information concerning protein secondary structure than can be meaningfully interpreted. This is due in part to limited models for secondary structure. Using the algorithm described in the previous paper of this series, we have generated a library of substructures for several trypsin-like serine proteases. This library was used as a basis for spectra-structure correlations with infrared spectra in the amide I' region, for five homologous proteins for which spectra were collected. Use of the substructure library has allowed correlations not previously possible with template-based methods of protein conformational analysis.


MD simulation of subtilisin BPN' in a crystal environment.

In this paper we present a molecular dynamics (MD) simulation of subtilisin BPN' in a crystalline environment containing four protein molecules and solvent. Conformational and dynamic properties of the molecules are compared with each other and with respect to the X-ray structure to test the validity of the force field. The agreement between simulated and experimental structure using the GROMOS force field is better than that obtained in the literature using other force fields for protein crystals. The overall shape of the molecule is well preserved, as is the conformation of alpha-helices and beta-strands. Structural differences are mainly found in loop regions. Solvent networks found in the X-ray structure were reproduced by the simulation, which was unbiased with respect to the crystalline hydration structure. These networks seem to play an important role in the stability of the protein; evidence of this is found in the structure of the active site. The weak ion binding site in the X-ray structure of subtilisin BPN' is occupied by a monovalent ion. When a calcium ion is placed in the initial structure, three peptide ligands are replaced by 5 water ligands, whereas a potassium ion retains (in part) its original ligands. Existing force fields yield a reliable method to probe local structure and short-time dynamics of proteins, providing an accuracy of about 0.1 nm.


Application of a directed conformational search for generating 3-D coordinates for protein structures from alpha-carbon coordinates.

A directed conformational search algorithm using the program CONGEN (ref. 3), which samples backbone conformers, is described. The search technique uses information from the partially built structures to direct the search process and is tested on the problem of generating a full set of backbone Cartesian coordinates given only alpha-carbon coordinates. The method has been tested on six proteins of known structure, varying in size and classification, and was able to generate the original backbone coordinates with RMSs ranging from 0.30-0.87A for the alpha-carbons and 0.5-0.99A RMSs for the backbone atoms. Cis peptide linkages were also correctly identified. The procedure was also applied to two proteins available with only alpha-carbon coordinates in the Brookhaven Protein Data Bank; thioredoxin (SRX) and triacylglycerol acylhydrolase (TGL). All-atom models are proposed for the backbone of both these proteins. In addition, the technique was applied to randomized coordinates of flavodoxin to assess the effects of irregularities in the data on the final RMS. This study represents the first time a deterministic conformational search was used on such a large scale.


Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I.

The function of the cellulose-binding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site-directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain. The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose. Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge-shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD. However, there was no effect on the activity toward small oligosaccharide (4-methylumbelliferyl beta-D-lactoside). The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important. However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants. The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI. These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose.


Modeling the anti-CEA antibody combining site by homology and conformational search.

A model for an antibody specific for the carcinoembryonic antigen (CEA) has been constructed using a method which combines the concept of canonical structures with conformational search. A conformational search technique is introduced which couples random generation of backbone loop conformations to a simulated annealing method for assigning side chain conformations. This technique was used both to verify conformations selected from the set of known canonical structures and to explore conformations available to the H3 loop in CEA ab initio. Canonical structures are not available for H3 due to its variability in length, sequence, and observed conformation in known antibody structures. Analysis of the results of conformational search resulted in three equally probable conformations for H3 loop in CEA. Force field energies, solvation free energies, exposure of charged residues and burial of hydrophobic residues, and packing of hydrophobic residues at the base of the loop were used as selection criteria. The existence of three equally plausible structures may reflect the high degree of flexibility expected for an exposed loop of this length. The nature of the combining site and features which could be important to interaction with antigen are discussed.


Crystallization, sequence, and preliminary crystallographic data for an antipeptide Fab 50.1 and peptide complexes with the principal neutralizing determinant of HIV-1 gp120.

X-ray quality crystals of an Fab fragment from an antipeptide monoclonal antibody (R/V3-50.1) that recognizes the principal neutralizing determinant (PND) of the gp120 glycoprotein of human immunodeficiency virus type 1 (HIV-1) (MN isolate) were grown as uncomplexed and peptide complexed forms. Crystals of the free Fab grew from high salt in orthorhombic space groups P2(1)2(1)2(1) and I222 and from polyethylene glycol in space groups P1 and P2(1). Seeds from either the P1 and P2(1) native (uncomplexed) Fab crystals induced nucleation of crystals of the Fab complexed to a 16-residue synthetic peptide corresponding to the PND when streak seeded into preequilibrated solutions of this complex. Data were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 A resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding of the PND of HIV-1 and its recognition by neutralizing monoclonal antibodies.


Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library.

Methods have recently been developed to present vast libraries of random peptides on the surface of filamentous phage. To introduce a degree of conformational constraint into random peptides, a library of hexapeptides flanked by cysteine residues (capable of forming cyclic disulfides) was constructed. This library was screened using the platelet glycoprotein, IIb/IIIa, which mediates the aggregation of platelets through binding of fibrinogen. A variety of peptides containing the sequence Arg-Gly-Asp or Lys-Gly-Asp were discovered and synthesized. The cyclic, disulfide-bonded forms of the peptides bound IIb/IIIa with dissociation constants in the nanomolar range, while reduced forms or an analogue in which Ser replaced the Cys residues bound considerably less tightly. These results demonstrate the feasibility for introducing conformational constraints into random peptide libraries and also demonstrates the potential for using phage peptide libraries to discover pharmacologically active lead compounds.


Binding of sequence-specific proteins to the adenosine- plus uridine-rich sequences of the murine granulocyte/macrophage colony-stimulating factor mRNA.

Adenosine+uridine (AU)-rich sequences in the 3' untranslated region (3'UTR) of the mRNA of many cytokines and oncogenes play an important role in mediating RNA degradation. Among the cytokines containing such AU-rich sequences in their 3'UTR is the hematopoietic growth factor granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF gene expression in T cells is regulated by modulation of mRNA half-life. Transfection studies using murine EL-4 thymoma cells have demonstrated that degradation depends on the presence of specific elements in the 3'UTR, including the AU-rich sequences. A number of AU-binding factors have recently been discovered, suggesting that specific regulation may occur through specific protein-mRNA interaction(s). We present evidence from gel-shift analyses and label-transfer experiments that murine cells contain proteins that bind specifically to AU-rich sequences. Three major proteins of 33, 39.5, and 42 kDa are detected. Phorbol ester treatment of cells does not alter the abundance or apparent binding affinity of the proteins. The 33-kDa protein is present in the cytoplasm of murine and human cells, whereas the 39.5- and 42-kDa proteins are present in murine extracts only. Constitutively expressed AU-binding proteins of the type that we describe may function by directing mRNA degradation in the absence of a stimulus to the contrary.


Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes.

Successful implementation of bone marrow transplantation for hematopoietic reconstitution is limited by the lack of suitably HLA-matched donors and by the occurrence of graft-versus-host disease that frequently accompanies this procedure. Recent clinical reports have implied that the use of umbilical cord blood as a source of transplantable stem cells may solve these problems. To date, definitive experiments have not been performed to assess the immunological potential of T cells found in umbilical cord blood, which could mediate graft-versus-host disease. In the present study we have observed that umbilical cord blood contains T lymphocytes that appear to be phenotypically immature. In addition, umbilical cord blood lymphocytes appeared to be functionally immature as shown by minimal responses to stimulation with interleukin 2, phytohemagglutinin, or alloantigens. Thus, umbilical cord blood may be more suitable for allogeneic transplantation than bone marrow in that these cord blood cells may not be as capable of mediating graft-versus-host disease.


Complement activation by beta-amyloid in Alzheimer disease.

Alzheimer disease (AD) is characterized by excessive deposition of the beta-amyloid peptide (beta-AP) in the central nervous system. Although several lines of evidence suggest that beta-AP is neurotoxic, a mechanism for beta-AP toxicity in AD brain remains unclear. In this paper we provide both direct in vitro evidence that beta-AP can bind and activate the classical complement cytolytic pathway in the absence of antibody and indirect in situ evidence that such actions occur in the AD brain in association with areas of AD pathology.


A binary plasmid system for shuffling combinatorial antibody libraries.

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis.


Hepatitis B virus capsid particles are assembled from core-protein dimer precursors.

Our studies on the assembly of hepatitis B virus capsids or core particles in Xenopus oocytes have demonstrated that unassembled p21.5 core proteins ("free p21.5") provide a pool of low-molecular-mass precursors for core-particle assembly. Here we have characterized this material. Free p21.5 sedimented through gradients of 3-25% sucrose (wt/vol) as a single protein species of approximately 40 kDa, corresponding to a p21.5 dimer. On nonreducing SDS/polyacrylamide gels, free p21.5 migrated as disulfide-linked p21.5 dimeric species of 35 and 37 kDa. Truncated core proteins lacking most or all of the 36-amino acid protamine region at the p21.5 carboxyl terminus were also found to behave as disulfide-linked dimers with appropriately reduced molecular masses. Our experiments failed to reveal monomeric core proteins or stable intermediates between dimers and capsids along the assembly pathway. We conclude that hepatitis B virus core particles are most likely assembled by aggregating 90 (or possibly 180) disulfide-linked p21.5 dimers. We discuss similarities between the assembly of hepatitis B virus capsids and simple T = 3 plant virus and bacteriophage structures.


Retinoic acid is enriched in Hensen's node and is developmentally regulated in the early chicken embryo.

Retinoic acid (RA) has been considered as a potential morphogen in the chicken limb and has also been suggested to be involved in early embryonic development. On the basis of biological activity, previous reports suggest that Hensen's node, the anatomical equivalent in the chicken of the Spemann's organizer, may contain RA. Here, by using a molecular assay system, we demonstrate that Hensen's node contains retinoids in a concentration approximately 20 times more than that in the neighboring tissues. Furthermore, stage 6 Hensen's node contains approximately 3 times more retinoid than that of stage 4 embryos. These endogenous retinoids may establish a concentration gradient from Hensen's node to adjacent tissues and play a role in establishing the primary embryonic axis in the vertebrate. The results also suggest that the retinoid concentration in Hensen's node is developmentally regulated.


Large, identical, tandem repeating units in the C protein alpha antigen gene, bca, of group B streptococci.

Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C protein alpha antigen of GBS is thought to have a role in both virulence and immunity. We previously cloned the C protein alpha antigen structural gene (named bca for group B, C protein, alpha) into Escherichia coli. Western blots of both the native alpha antigen and the cloned gene product demonstrate a regularly laddered pattern of heterogeneous polypeptides. The nucleotide sequence of the bca locus reveals an open reading frame of 3060 nucleotides encoding a precursor protein of 108,705 Da. Cleavage of a putative signal sequence of 41 amino acids yields a mature protein of 104,106 Da. The 20,417-Da N-terminal region of the alpha antigen shows no homology to previously described protein sequences and is followed by a series of nine tandem repeating units that make up 74% of the mature protein. Each repeating unit is identical and consists of 82 amino acids with a molecular mass of 8665 Da, which is encoded by 246 nucleotides. The size of the repeating units corresponds to the observed size differences in the heterogeneous ladder of alpha C proteins expressed by GBS. The C-terminal region of the alpha antigen contains a membrane anchor domain motif that is shared by a number of Gram-positive surface proteins. The large region of identical repeating units in bca defines protective epitopes and may play a role in generating phenotypic and genotypic diversity of the alpha antigen.


Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes.

Survival of Salmonella typhimurium within macrophage phagosomes requires the coordinate expression of bacterial gene products. This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S. typhimurium virulence regulators PhoP and PhoQ. Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold. In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage phagosomes. The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments. Measurements of pH documented that S. typhimurium delayed and attenuated acidification of its intracellular compartment. Phagosomes containing S. typhimurium required 4-5 hr to reach pH < 5.0. In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5. The eventual acidification of phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression. These observations implicate phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate phagosome acidification.


Retention of habituation in PC12 cells.

The time course of habituation and recovery of neurotransmitter release was measured in neuronally differentiated PC12 cells stimulated with either acetylcholine or ATP. The release of norepinephrine in response to either stimulant declined exponentially with repeated presentation of that stimulant. When the stimulus was withheld, the cells' ability to respond recovered to initial levels with an exponential time course. The rate of response recovery depended on the stimulant used and, in the case of stimulation with acetylcholine, on the number of previous stimuli. After habituation and recovery, or partial recovery, of norepinephrine release, the response to a second series of repetitive stimuli declined more rapidly than in the naive case. This increase in habituation rate was dependent on the number of previous stimuli and, in the case of stimulation with acetylcholine, was stable with time for at least 90 min after stimulation. These phenomena are analogous to characteristics of short- and long-term memories of habituative learning observed in behavioral studies. Kinetic equations based on a putative reversible stimulation-dependent inactivation of the cellular response mechanism were used to analyze the rates of habituation and response recovery.


Structural analysis of ternary complexes of vaccinia RNA polymerase.

The structure of the elongation complex of vaccinia RNA polymerase halted at discrete template positions was examined by DNase I footprinting. The leading edge of the footprint bore a constant relationship to the catalytic template position, being 22-24 nucleotides (nt) in advance on the nontemplate strand and 17 nt on the template strand. DNase hypersensitivity of the nontemplate strand at the leading edge suggested that the DNA might be distorted as it entered the polymerase molecule. The region of DNA unwinding at the transcription bubble extended at least 12 nt 5' from the catalytic center, as indicated by the reactivity of adenosine residues to diethylpyrocarbonate. Cu-phenanthroline-hypersensitive sites located 13 nt 5' and 4 nt 3' of the growing point appeared to demarcate the margins of the bubble. Strand asymmetry of chemical modification within the bubble was consistent with an RNA-DNA hybrid of no more than 10 base pairs.


Telomere length predicts replicative capacity of human fibroblasts.

When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explained by the chronological age of the donor. Because we had previously shown that telomeres, the terminal guanine-rich sequences of chromosomes, shorten throughout the life-span of cultured cells, we wished to determine whether variation in initial telomere length would account for the unexplained variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93 yr) indicated relatively weak correlations between proliferative ability and donor age (m = -0.2 doubling per yr; r = -0.42; P = 0.02) and between telomeric DNA and donor age (m = -15 base pairs per yr; r = -0.43; P = 0.02). However, there was a striking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublings per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent significantly fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker of somatic cell aging in humans and are consistent with a causal role for telomere loss in this process. We also found that fibroblasts from Hutchinson-Gilford progeria donors had short telomeres, consistent with their reduced division potential in vitro. In contrast, telomeres from sperm DNA did not decrease with age of the donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.


Comparison of the NMR solution structure and the x-ray crystal structure of rat metallothionein-2.

Metallothioneins are small cysteine-rich proteins capable of binding heavy metal ions such as Zn2+ and Cd2+. They are ubiquitous tissue components in higher organisms, which tentatively have been attributed both unspecific protective functions against toxic metal ions and highly specific roles in fundamental zinc-regulated cellular processes. In this paper a detailed comparison of the NMR solution structure [Schultze, P., Wörgötter, E., Braun, W., Wagner, G., Vasák, M., Kägi, J. H. R. & Wüthrich, K. (1988) J. Mol. Biol. 203, 251-268] and a recent x-ray crystal structure [Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xoung, N. H., Furey, W. F., Wang, B. C. & Stout, C. D. (1991) J. Mol. Biol. 221, 1269-1293] of rat metallothionein-2 shows that the metallothionein structures in crystals and in solution have identical molecular architectures. The structures obtained with both techniques now present a reliable basis for discussions on structure-function correlations in this class of metalloproteins.


Immobilization of DNA for scanning probe microscopy.

Reproducible scanning tunneling microscope and atomic force microscope images of entire molecules of uncoated plasmid DNA chemically bound to surfaces are presented. The chemically mediated immobilization of DNA to surfaces and subsequent scanning tunneling microscope imaging of DNA molecules demonstrate that the problem of molecular instability to forces exerted by the probe tip, inherent with scanning probe microscopes, can be prevented.


The same exhaustible multilineage precursor produces both myeloid and lymphoid cells as early as 3-4 weeks after marrow transplantation.

Hemopoietic precursors with the ability to differentiate into wide varieties of cell types are considered primitive, as are precursors with long-term repopulating ability. Here we study the populations of marrow precursors from which both myeloid and lymphoid lineages are descended shortly after transplantation. Surprisingly, few or none of these precursors show long-term repopulating ability. Equal portions of a mixture of marrow cells from C57BL/6J (B6) and congenic B6-Hbbd Gpi-1a mice are transplanted into a group of recipients. Three weeks later, highly significant correlations between percentages of B6 type T cells, B cells, granulocytes, and platelets in each recipient indicate that many lymphoid and myeloid cells are descended from common precursors. After 4-6 weeks, most correlations between lymphoid and myeloid cells improve, indicating that most or all differentiated cells are descended from common precursors. The more differentiated myeloid-specific precursors found in spleen colony-forming cell assays apparently fail to contribute significantly to the differentiated myeloid cell populations tested. By using the binomial model, in which variability of the data among the recipients is inversely related to the number of precursors in the mixture, donor precursor concentrations are estimated as approximately 21 per 10(5) marrow cells after 3 weeks, falling 3-fold to 6.6 per 10(5) after 4-6 weeks. This trend continues, with higher correlations, greater variabilities, and donor precursor concentrations of 1.9 per 10(5) marrow cells after 12-14 weeks and 1.4 per 10(5) after 24 weeks. Strong increases in variances between 3 and 12 weeks after transplantation suggest that most or all of the initially active multilineage precursors are exhausted during this time period. The fact that the ability of a hemopoietic stem cell to differentiate into widely disparate lineages is not associated with long-term repopulating ability requires a change in stem cell definitions, since primitive hemopoietic stem cells have traditionally been defined by both these abilities.


Structure of the ColE1 DNA molecule before segregation to daughter molecules.

The segregation of daughter DNA molecules at the end stage of replication of plasmid ColE1 was examined. When circular ColE1 DNA replicates in a cell extract at a high KCl concentration (140 mM), a unique class of molecules accumulates. When the molecule is cleaved by a restriction enzyme that cuts the ColE1 DNA at a single site, an X-shaped molecule in which two linear components are held together around the origin of DNA replication is made. For a large fraction of these molecules, the 5' end of the leading strand remains at the origin and the 3' end of the strand is about 30 nucleotides upstream of the origin. The 3' end of the lagging strand is located at the terH site (17 nucleotides upstream of the origin) and the 5' end of the strand is a few hundred nucleotides upstream of the terH site. Thus the parental strands of the molecule intertwine with each other only once. When the KCl concentration is lowered to 70 mM, practically all of these molecules are converted to daughter circular monomers or to catenanes consisting of two singly interlocked circular units.


Time-resolved circularly polarized protein phosphorescence.

The existence of circular polarization in room-temperature protein phosphorescence is demonstrated, and time-resolved circularly polarized phosphorescence (TR-CPP) is used to characterize unique tryptophan environments in multitryptophan proteins. Circularly polarized luminescence studies provide information regarding the excited state chirality of a lumiphore which can be used to extract sensitive structural information. It is shown by time resolving the circular polarization that it is possible to correlate the excited state chirality with unique decay components in a multiexponential phosphorescence decay profile. The present study presents a concurrent analysis of room-temperature time-resolved phosphorescence and TR-CPP of bacterial glucose-6-phosphate dehydrogenase as well as those of horse liver alcohol dehydrogenase. Only one of the two tryptophan residues per subunit of dimeric alcohol dehydrogenase is believed to phosphorescence, while the dimeric glucose-6-phosphate dehydrogenase has eight tryptophan residues per subunit and shows a corresponding complexity in its phosphorescence decay profile. The anisotropy factor [g(em) = delta I/(Itotal/2); delta I = Ileft circular-Iright circular] for alcohol dehydrogenase is time independent, suggesting a unique excited state chirality. The phosphorescence decay of glucose-6-phosphate dehydrogenase can be well fitted with four exponential terms of 4, 23, 76, and 142 msec, and the TR-CPP of this enzyme shows a strong time dependence that can be resolved into four individual time-independent anisotropy factors of -4.0, -2.1, +6.5, and +6.9 (x10(-3)), each respectively associated with one of the four lifetime components. These results demonstrate how the use of TR-CPP can facilitate the study of proteins with multiple lumiphores.


Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies.

Several lines of CHO cells stably overexpressing protein kinase C (PKC) subspecies to various extents were established by the DNA-mediated transfer. Upon treatment with phorbol 12-myristate 13-acetate, the growth of the cells expressing the PKC-delta subspecies was markedly inhibited, whereas cell lines expressing PKC-alpha, PKC-beta II, and PKC-zeta subspecies were not significantly affected. Flow cytometric analysis indicated that all cell lines overexpressing PKC-delta subspecies accumulated in G2/M phase in response to phorbol 12-myristate 13-acetate. In these arrested cells, dikaryons were predominant, implying that phorbol ester-induced inhibition of cell division is specific to telophase. These results suggest PKC-delta subspecies may play a role in the normal cell cycle progression.


Ser-752-->Pro mutation in the cytoplasmic domain of integrin beta 3 subunit and defective activation of platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia.

Integrins are membrane receptors which mediate cell-cell or cell-matrix adhesion. Integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) acts as a fibrinogen receptor of platelets and mediates platelet aggregation. Platelet activation is required for alpha IIb beta 3 to shift from noncompetent to competent for binding soluble fibrinogen. The steps involved in this transition are poorly understood. We have studied a variant of Glanzmann thrombasthenia, a congenital bleeding disorder characterized by absence of platelet aggregation and fibrinogen binding. The patient's platelets did not bind fibrinogen after platelet activation by ADP or thrombin, though his platelets contained alpha IIb beta 3. However, isolated alpha IIb beta 3 was able to bind to an Arg-Gly-Asp-Ser affinity column, and binding of soluble fibrinogen to the patient's platelets could be triggered by modulators of alpha IIb beta 3 conformation such as the Arg-Gly-Asp-Ser peptide and alpha-chymotrypsin. These data suggested that a functional Arg-Gly-Asp binding site was present within alpha IIb beta 3 and that the patient's defect was not secondary to a blockade of alpha IIb beta 3 in a noncompetent conformational state. This was evocative of a defect in the coupling between platelet activation and alpha IIb beta 3 up-regulation. We therefore sequenced the cytoplasmic domain of beta 3, following polymerase chain reaction (PCR) on platelet RNA, and found a T-->C mutation at nucleotide 2259, corresponding to a Ser-752-->Pro substitution. This mutation is likely to be responsible for the uncoupling of alpha IIb beta 3 from cellular activation because (i) it is not a polymorphism, (ii) it is the only mutation in the entire alpha IIb beta 3 sequence, and (iii) genetic analysis of the family showed that absence of the Pro-752 beta 3 allele was associated with the normal phenotype. Our data thus identify the C-terminal portion of the cytoplasmic domain of beta 3 as an intrinsic element in the coupling between alpha IIb beta 3 and platelet activation.


Isolation of a wheat cDNA clone for an abscisic acid-inducible transcript with homology to protein kinases.

Increases in the plant hormone abscisic acid (ABA) initiate water-stress responses in plants. We present evidence that a transcript with homology to protein kinases is induced by ABA and dehydration in wheat. A 1.2-kilobase cDNA clone (PKABA1) was isolated from an ABA-treated wheat embryo cDNA library by screening the library with a probe developed by polymerase chain reaction amplification of serine/threonine protein kinase subdomains VIb to VIII. The deduced amino acid sequence of the PKABA1 clone contains the features of serine/threonine protein kinases, including homology with all 12 conserved regions of the catalytic domain. PKABA1 transcript levels are barely detectable in growing seedlings but are induced dramatically when plants are subjected to dehydration stress. The PKABA1 transcript can also be induced by supplying low concentrations of ABA, and coordinate increases in ABA levels and PKABA1 mRNA occur when seedlings are water-stressed. Identification of this ABA-inducible transcript with homology to protein kinases provides a basis for examining the role of protein phosphorylation in plant responses to dehydration.


Cloning and expression of an NADP(+)-dependent alcohol dehydrogenase gene of Entamoeba histolytica.

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entamoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoelectric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP(+)-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADH1 clone had an open reading frame that was 360 amino acids long and encoded a protein of approximately 39 kDa (calculated size). EhADH1 showed a 62% amino acid identity with the tetrameric NADP(+)-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADH1 showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADH1 contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADH1 lacked a 23-amino acid stretch present in other alcohol dehydrogenases that includes four cysteines that bind a second noncatalytic zinc ion. An EhADH1-glutathione-S-transferase fusion protein showed the expected NADP(+)-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADH1 fusion protein were inhibited by pyrazole and 4-methylpyrazole.


Homocysteine and other sulfhydryl compounds enhance the binding of lipoprotein(a) to fibrin: a potential biochemical link between thrombosis, atherogenesis, and sulfhydryl compound metabolism.

We have previously shown that lipoprotein(a) [Lp(a)], an atherogenic lipoprotein that contains apolipoprotein(a), which shares partial structural homology to plasminogen, binds to a plasmin-modified fibrin surface, and we have postulated that this interaction may be atherogenic. Moderate elevations in blood homocysteine, a relatively common condition, predispose to premature atherosclerosis. The reasons for this are not established. We now report that homocysteine, at concentrations as low as 8 microM, significantly increases the affinity of Lp(a) for fibrin. Homocysteine induces a 20-fold increase in the affinity between Lp(a) and plasmin-treated fibrin and a 4-fold increase with unmodified fibrin. Lp(a) binding is inhibited by epsilon-aminocaproic acid, indicating lysine binding site specificity. Homocysteine does not enhance the binding of Lp(a) to other surface-bound proteins. Cysteine, glutathione, and N-acetylcysteine also increase the affinity between Lp(a) and fibrin. Homocysteine does not affect the binding of low density lipoprotein or plasminogen to fibrin, nor does it alter the gel-filtration elution pattern of Lp(a). Immunoblot analysis documents the fact that homocysteine partially reduces Lp(a). These results suggest that homocysteine alters the intact Lp(a) particle so as to increase the reactivity of the plasminogen-like apolipoprotein(a) portion of the molecule. The observation that sulfhydryl amino acids increase Lp(a) binding to fibrin suggests a biochemical relationship between sulfhydryl compound metabolism, thrombosis, and atherogenesis.


Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein.

Infectious RNA transcripts were generated from full-length cDNA clones of the tobacco etch potyvirus genome containing an insertion of the bacterial beta-glucuronidase (GUS) gene between the polyprotein-coding sequences for the N-terminal 35-kDa proteinase and the helper component-proteinase. The recombinant virus was able to spread systemically in plants and accumulated to a level comparable with wild-type tobacco etch potyvirus. Proteolytic processing mediated by the 35-kDa proteinase and helper component-proteinase resulted in production of an enzymatically active GUS-helper component-proteinase fusion protein. A virus passage line that retained the GUS insert after numerous plant-to-plant transfers, as well as a line that sustained a deletion of the GUS sequence, was recovered. Use of an in situ histochemical GUS assay in time-course experiments allowed the visualization of virus activity in single, mechanically inoculated leaf epidermal cells, in neighboring epidermal and mesophyll cells, in phloem-associated cells after long-distance transport, and in cells surrounding vascular tissues of organs above and below the site of inoculation. This system represents a powerful tool to study plant virus replication, short- and long-distance virus movement, and virus-host interactions. Additionally, we show that potyviruses may serve as highly efficient, autonomously replicating vectors for the expression of foreign genes in plants.


CD22-mediated stimulation of T cells regulates T-cell receptor/CD3-induced signaling.

Interaction between B lymphocytes and other cell types is mediated in part by the B-cell adhesion molecule CD22. Recent work has suggested one of the T-cell ligands of B cells to be CD45RO, an isoform of the receptor-linked phosphotyrosine phosphatase CD45. Here we demonstrate direct interaction between CD22 and several isoforms of CD45, including CD45RO, and propose that the interaction may participate in regulation of lymphocyte signaling. Cross-linking of CD3 and CD22 T-cell ligands with anti-CD3 antibody and soluble CD22 is shown to block anti-CD3-induced intracellular calcium increase and to inhibit tyrosine phosphorylation of phospholipase C gamma 1. These effects are consistent with those observed upon coligation of CD3 and CD45 with antibody, providing support to the possibility that ligand-mediated stimulation of CD45 may result in modulation of substrate phosphorylation and lymphocyte activation.


Relationship of human immunodeficiency virus type 1 sequence heterogeneity to stage of disease.

V3 envelope sequences were determined from amplified human immunodeficiency virus type 1 (HIV-1) sequences of uncultivated leukocytes obtained sequentially from four infected adults over the course of infection. Lower levels of sequence heterogeneity were noted in samples obtained early in HIV-1 infection, prior to CD4 depletion, than in samples obtained at later times during disease. The pattern of amino acid sequence divergence included nonrandom changes, with evidence of sequence variants arising from HIV-1 quasi-species present earlier in infection. Consensus sequences for isolates obtained early after infection from different patients demonstrated a high level of conservation with one another and a consensus sequence for macrophage-tropic HIV-1 isolates. These findings suggest that a highly restricted subset of HIV-1 quasi-species can be transmitted and can establish infection.


A periplasmic protein disulfide oxidoreductase is required for transformation of Haemophilus influenzae Rd.

The mutated gene in JG16, a Haemophilus influenzae strain deficient in competence-induced DNA binding and uptake, was cloned and the wild-type allele was sequenced. The gene was shown by Northern analysis to be constitutively expressed on a 1.7-kilobase transcript. The gene product was identified as a 20.6-kDa protein targeted to the periplasm. The protein contains the sequence Cys-Pro-His-Cys (CPHC) and is highly similar to two other periplasmic CPHC motif-containing proteins: DsbA, an Escherichia coli protein (45% identity, 87% homology) and TcpG, a Vibrio cholerae protein (32% identity, 74% homology). Both DsbA and TcpG promote disulfide bond formation in periplasmic proteins, are required for pilus biogenesis, and, like thioredoxin, are capable of reducing insulin in vitro. The Haemophilus protein was shown to complement an E. coli mutation in DsbA and was named Por (periplasmic oxidoreductase). In JG16 the competence-dependent redistribution of inner membrane proteins did not occur. These findings suggest that Por is required for the correct assembly and/or folding of one or more disulfide-containing cell envelope protein involved either in competence development or in the DNA-binding and -uptake machinery.


Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin.

Proteolytic cleavage of the protective antigen (PA) protein of anthrax toxin at residues 164-167 is necessary for toxic activity. Cleavage by a cellular protease at this sequence, Arg-Lys-Lys-Arg, normally follows binding of PA to a cell surface receptor. We attempted to identify this protease by determining its sequence specificity and catalytic properties. Semi-random cassette mutagenesis was used to generate mutants with replacements of residues 164-167 by Arg, Lys, Ser, or Asn. Analysis of 19 mutant proteins suggested that lethal factor-dependent toxicity required the sequence Arg-Xaa-Xaa-Arg. Based on these data, three additional mutants were constructed with the sequences Ala-Lys-Lys-Arg, Arg-Lys-Lys-Ala, and Arg-Ala-Ala-Arg. Of these mutant proteins, Arg-Ala-Ala-Arg was toxic, confirming that the cellular protease can recognize the sequence Arg-Xaa-Xaa-Arg. The mutant containing the sequence Ala-Lys-Lys-Arg was also toxic but required > 13 times more protein to produce equivalent toxicity. This sequence specificity is similar to that of the ubiquitous subtilisin-like protease furin, which is involved in processing of precursors of certain receptors and growth factors. Therefore we tested whether a recombinant soluble furin would cleave PA. This furin derivative efficiently cleaved native PA and the Arg-Ala-Ala-Arg mutant but not the nontoxic PA mutants. In addition, previously identified inhibitors of furin blocked cleavage of receptor-bound PA. These data imply that furin is the cellular protease that activates PA, and that nearly all cell types contain at least a small amount of furin exposed on their cell surface.


A histidine protein kinase homologue required for regulation of bacterial cell division and differentiation.

Differentiation in the dimorphic bacterium Caulobacter crescentus results from a sequence of discontinuous, stage-specific events that leads to the production of a stalked cell and a new motile swarmer cell after each asymmetric cell division. As reported previously, pseudoreversion analysis of mutations in the pleiotropic developmental gene pleC identified three cell division genes: divJ, divK, and divL. We show here that one of these genes, divJ, encodes a predicted protein of 596 residues with an extensive hydrophobic N-terminal region and a C-terminal domain containing all of the invariant residues found in the family of bacterial histidine protein kinases. Our results also show that divJ is discontinuously transcribed early in the swarmer cell cycle during a period that coincides with the G1 to S transition. We propose that the DivJ protein is one member of a signal transduction pathway regulating the cell cycle and differentiation in Caulobacter and that protein modification by phosphorylation may play a central role in coupling developmental events to progress through the cell division cycle.


Somatic recombination of heavy chain variable region transgenes with the endogenous immunoglobulin heavy chain locus in mice.

Transgenic lines of mice were derived by using plasmid constructs containing DNA encoding an antibody heavy chain variable-diversity-joining region (VH-D-JH) and various amounts of 5' and 3' flanking DNA but lacking any repetitive isotype switch (S) or constant (C) region DNA. Unexpectedly, many of the antibody VH regions expressed by B-cell hybridomas generated from immunized transgenic mice were found to be of transgenic origin. Further analyses showed that somatic events had generated hybrid genomic loci in the mice containing the transgenic VH-D-JH gene and plasmid sequences 5' of endogenous heavy chain C region genes. Thus, VH-D-JH transgenes lacking S and C region DNA can recombine with endogenous Igh DNA, leading to the expression of transgene-encoded antibody.


Induction of the neural cell adhesion molecule and neuronal aggregation by osteogenic protein 1.

The neural cell adhesion molecule (N-CAM) plays a fundamental role in nervous system development and regeneration, yet the regulation of the expression of N-CAM in different brain regions has remained poorly understood. Osteogenic protein 1 (OP-1) is a member of the transforming growth factor beta superfamily that is expressed in the nervous system. Treatment of the neuroblastoma-glioma hybrid cell line NG108-15 for 1-4 days with recombinant human OP-1 (hOP-1) induced alterations in cell shape, formation of epithelioid sheets, and aggregation of cells into multilayered clusters. Immunofluorescence studies and Western blots demonstrated a striking differential induction of the three N-CAM isoforms in hOP-1-treated cells. hOP-1 caused a 6-fold up-regulation of the 140-kDa N-CAM, the isoform showing the highest constitutive expression, and a 29-fold up-regulation of the 180-kDa isoform. The 120-kDa isoform was not detected in control NG108-15 cells but was readily identified in hOP-1-treated cells. Incubation of NG108-15 cells with an antisense N-CAM oligonucleotide reduced the induction of N-CAM by hOP-1 and decreased the formation of multilayered cell aggregates. Anti-N-CAM monoclonal antibodies also diminished the formation of multilayered cell aggregates by hOP-1 and decreased cell-cell adhesion when hOP-1-treated NG108-15 cells were dispersed and replated. Thus, hOP-1 produces morphologic changes in NG108-15 cells, at least in part, by inducing N-CAM. These observations suggest that OP-1 or a homologue may participate in the regulation of N-CAM during nervous system development and regeneration.


Alternative splicing generates metabotropic glutamate receptors inducing different patterns of calcium release in Xenopus oocytes.

A splice variant of the metabotropic glutamate receptor (mGluR) 1a, named mGluR1c, was isolated. Compared to mGluR1a, the predicted mGluR1c protein is 302 amino acids shorter at its C-terminal end. Despite this difference, mGluR1c activates phospholipase C in Xenopus oocytes with a pharmacological profile identical to that of mGluR1a. However, in contrast to the large fast transient responses induced by mGluR1a, mGluR1c receptors elicit a small more slowly generated long-lasting oscillatory current, suggesting that these two receptors do not generate the same pattern of Ca2+ release in Xenopus oocytes. In situ hybridization data show that mGluR1c mRNA is expressed at a lower level than the other splice variants of mGluR1. Some differences in the regional distribution of these transcripts were observed in the cerebellum, the olfactory bulb, and the striatum.


Quadruplex structure of d(G3T4G3) stabilized by K+ or Na+ is an asymmetric hairpin dimer.

The ends of chromosomes contain repeats of guanine-rich sequences that can assume highly compact conformations and are presumed necessary for their biological role in chromosomal stabilization and association. We have investigated the conformational behavior of d(G3T4G3) as a function of the addition of either KCl or NaCl, in the concentration range of 50-200 mM, by using a spectrum of physical techniques and conclude that these salts induce a quadruplex species composed of two strands, each in a hairpin conformation. When salt is added, a large positive signal appears near 290 nm in the CD spectra. UV thermal denaturation curves show a single concentration-dependent transition and provide data for quantitating the thermodynamics of quadruplex formation. In electrophoresis experiments, the quadruplex structure migrates as a single species and more rapidly than the unstructured single strand. NMR spectra in the presence of KCl or NaCl indicate that the structure formed is asymmetric. Equilibrium ultracentrifugation studies confirm that these quadruplexes are composed of two strands of d(G3T4G3). Possible models for this structure are discussed.


DnaJ, DnaK, and GrpE heat shock proteins are required in oriP1 DNA replication solely at the RepA monomerization step.

We have found that three Escherichia coli heat shock proteins, DnaK (the hsp70 homolog), DnaJ, and GrpE, function in oriP1 DNA replication in vitro solely to activate DNA binding by the replication initiator protein RepA. Activation results from the conversion of P1 or P7 RepA dimers to monomers that bind with high affinity to the origin of replication of plasmid P1. Thus, the essential role of these three heat shock proteins in this replication system is to change the quaternary structure of a single protein, RepA.


Adaptive cellular interactions in the immune system: the tunable activation threshold and the significance of subthreshold responses.

A major challenge for immunologists is to explain how the immune system adjusts its responses to the microenvironmental context in which antigens are recognized. We propose that lymphocytes achieve this by tuning and updating their responsiveness to recurrent signals. In particular, cellular anergy in vivo is a dynamic state in which the threshold for a stereotypic mode of activation has been elevated. Anergy is associated with other forms of cellular activity, not paralysis. Cells engaged in such subthreshold interactions mediate functions such as maintenance of immunological memory and control of infections. In such interactions, patterns of signals are recognized and classified and evoke selective responses. The robust mechanism proposed for segregation of suprathreshold and subthreshold immune responses allows lymphocytes to use recognition of self-antigens in executing physiological functions. Autoreactivity is allowed where it is dissociated from uncontrolled aggression.


Uptake of exogenous free cholesterol induces upregulation of tissue factor expression in human monocyte-derived macrophages.

Lipid-laden macrophages present as foam cells may contribute to the hyperthrombotic state of human atherosclerotic lesions by the production of tissue factor (TF). We investigated the effect of exogenous nonlipoprotein cholesterol on the expression of TF by human monocyte-derived macrophages in culture. Nonlipoprotein cholesterol at 50 micrograms/ml increased TF activity 4-fold; TF induction was dose- and time-dependent. Expression of TF activity was positively correlated with the free cholesterol content of monocyte-derived macrophages, was increased upon inhibition of cholesterol esterification, and reflected de novo synthesis of TF protein. TF expression in cholesterol-loaded macrophages remained sensitive to stimulation (approximately 12-fold) by bacterial lipopolysaccharide, indicating that intracellular free cholesterol and lipopolysaccharide act by distinct mechanisms in inducing TF procoagulant activity. Our results suggest that loading human monocyte-derived macrophages with free cholesterol induces upregulation of TF expression, thereby contributing to thrombus formation at sites of plaque rupture.


Macrophages in Drosophila embryos and L2 cells exhibit scavenger receptor-mediated endocytosis.

Mammalian macrophage scavenger receptors exhibit unusually broad binding specificity and are implicated in atherosclerosis and host defense. Scavenger receptor-like endocytosis was observed in Drosophila melanogaster embryos and in primary embryonic cell cultures. This receptor activity was expressed primarily by macrophages. The Drosophila Schneider L2, but not the Kc, cell line also exhibited a scavenger receptor-mediated endocytic pathway similar to its mammalian counterpart. L2 receptors mediated high-affinity internalization and subsequent temperature- and chloroquine-sensitive degradation of 125I-labeled acetylated low density lipoprotein and displayed characteristic ligand specificity. These findings suggest that scavenger receptors mediate important, well-conserved functions and raise the possibility that they may be pattern recognition receptors that arose early in the evolution of host defense mechanisms. They also establish additional systems for the investigation of endocytosis in Drosophila and scavenger receptor function in disease, host defense, and development.


Identification of the gene for an Escherichia coli poly(A) polymerase.

Many bacterial mRNAs, like those of eukaryotes, carry a polyadenylate sequence at their 3' termini, but neither the function of the bacterial poly(A) moieties nor their biosynthesis have been elucidated. To develop a genetic tool to approach the problem of bacterial poly(A) RNA, we have sought to identify the genes responsible for mRNA polyadenylylation. A poly(A) polymerase was purified to homogeneity from extracts of Escherichia coli and subjected to N-terminal sequence analysis. The 25-residue amino acid sequence obtained was used to design primers for the amplification of the corresponding coding region by the PCR from an E. coli DNA template. A 74-base-pair DNA segment was obtained that matched a region in the pcnB locus of E. coli, a gene that had originally been identified as controlling plasmid copy number [J. Lopilato, S. Bortner & J. Beckwith (1986) Mol. Gen. Genet. 205, 285-290] and was subsequently cloned and sequenced [J. Liu & J. S. Parkinson (1989) J. Bacteriol. 171, 1254-1261]. Direct evidence that the pcnB locus encodes poly(A) polymerase was provided by the observation that a bacterial strain transformed with an inducible expression vector carrying pcnB as a translational fusion produced 100-fold elevated levels of poly(A) polymerase upon induction. No increased poly(A) polymerase activity was observed in cells transformed with expression vectors carrying truncated forms of the pcnB gene. The identification of a gene encoding bacterial poly(A) polymerase opens the way for the study of the biosynthesis and function of bacterial polyadenylylated mRNA.


Depth, motion, and static-flow perception at metaisoluminant color contrast.

Many experiments concerned with the role of color in depth and motion perception have applied isoluminant random-dot stereograms and cinematograms. The poor performance in the absence of luminance contrast has been associated with color-blindness of stereopsis and motion perception (Livingstone, M.S. & Hubel, D.H. (1987) J. Neurosci. 7, 3416-3468). Nevertheless, isoluminant stimuli are not fully accepted as appropriate tools in isolating central mechanisms (Logothetis, N.K., Schiller, P.H., Charles, E.R. & Hurlbert, A.C. (1990) Science 247, 214-217). In our experiments we use a broad luminance range to test whether color can contribute to a given mechanism when luminance contrast is present but has a strong "veto" effect from opposite luminance contrast, a condition we named "metaisoluminance." There is no fusion in stereopsis under polarity reversal, when only luminance information is given, and reversed-phi phenomenon is experienced for motion. As a third "matching" task, we included polarity-reversed random-dot Glass-patterns, which exhibit "static flow" and also show pattern reversal. We found that color can counteract the effects of polarity reversal by restoring stereoscopic fusion and reversed phi motion and does it with increased efficiency as the hue contrast increases. We found no such effect of color in Glass-patterns. Thus, we showed that the visual system for binocular depth and motion perception is not color-blind, although correlated hue information under metaisoluminance does not appear to yield shape perception.


Obliterative bronchiolitis after lung transplantation: a fibroproliferative disorder associated with platelet-derived growth factor.

Fibroproliferative disorders are characterized by accumulations of mesenchymal cells and connective tissue in critical locations, leading to organ dysfunction. We examined the role of platelet-derived growth factor (PDGF) in the pathogenesis of obliterative bronchiolitis, a fibroproliferative process that occurs after lung transplantation and results in small airway occlusion. Bronchoalveolar lavage fluid from obliterative bronchiolitis patients significantly stimulated fibroblast migration, whereas fluid from patient controls did not. Quantitation by radioligand binding assay demonstrated increased concentrations of PDGF in lavage fluid from obliterative bronchiolitis patients (patients, 104 +/- 26.9 pM; controls, 8.4 +/- 6.9 pM; P < 0.01). Heparin affinity, gel filtration, and Western blot analysis confirmed the presence of PDGF in lavage fluid. Immunohistochemical and in situ hybridization studies of histologic sections and bronchoalveolar lavage cells suggest that alveolar macrophages are one cellular source. Prospective evaluation of sequential bronchoalveolar lavage samples from a patient who developed obliterative bronchiolitis demonstrated markedly increased PDGF concentrations before the onset of irreversible airflow obstruction. These findings are consistent with a role for PDGF in the fibroproliferative changes observed in obliterative bronchiolitis.


Transplantation tolerance is unrelated to superantigen-dependent deletion and anergy.

C57BL/6 (B6; I-E-, Mls-2b) nude mice, reconstituted at birth with thymic epithelium (TE) from BALB/c (BA; I-E+, Mls-2a) day 10 embryos (E10), permanently accepted BALB/c skin, when grafted as adults. T-cell receptor repertoire analyses in the periphery of these mice revealed no difference in frequencies of I-E/superantigen-reactive T-cell receptor V beta families, as compared to chimeras constructed with syngeneic B6 E10 TE. T lymphocytes bearing V beta 3, V beta 5, and V beta 11 T-cell receptors, from either allogeneic or syngeneic TE chimeras, responded equally well to in vitro receptor-dependent stimulation. Similar results were obtained with nude mice reconstituted at birth with E14 thymuses, already colonized by hemopoietic cells. These observations indicate that neither TE cells nor the progenies of hemopoietic precursors that colonize the thymus up to E14 express or functionally present the superantigens addressed here; it follows that tolerance to skin grafts and superantigen-related T-cell deletions are unrelated phenomena.


Regulation of BALB/c 3T3 fibroblast proliferation by B-myb is accompanied by selective activation of cdc2 and cyclin D1 expression.

The B-myb gene is expressed in many cell types at the G1/S transition of the cell cycle. Inhibition of B-myb expression in BALB/c 3T3 fibroblasts by introduction of a B-myb antisense construct greatly diminished cell proliferation, whereas constitutive expression of a human B-myb cDNA in these cells reduced their growth factor requirements and induced a transformed phenotype. Constitutive expression of B-myb cDNA was accompanied by activation of cyclin D1 and cdc2 expression but not of cyclin A and cyclin B. Transfection of BALB/B-myb cells (a cell line expressing high levels of exogenous human B-myb) with a cyclin D1 antisense construct drastically reduced cloning efficiency of these cells. These results suggest that the B-myb-encoded product regulates fibroblast proliferation by activating cdc2 and cyclin D1 gene expression and that abnormal expression of cyclin D1 might be a step in the process of transformation.


Cloning and sequencing of a trophoblast-endothelial-activated lymphocyte surface protein: cDNA sequence and genomic structure.

We have previously described the distribution of a surface glycoprotein recognized by monoclonal antibody B721. We now report the molecular characterization of this molecule at the protein, cDNA, and genomic level. A 75-kDa glycoprotein can be immunoprecipitated from B721+ tissues. We have isolated a near full-length cDNA containing the complete coding region and a full-length genomic clone. We present evidence that this protein has similarity to several classes of nuclear transcription factors, particularly the myc family of proteins. The 721P protein was found to have a leucine zipper-like structure, a possible basic region immediately upstream from the leucine zipper, and a protein kinase A phosphorylation site. 721P protein is encoded by a gene distinct from any deposited in existing data bases, and displays several features associated with proteins involved in signal transduction and gene regulation.


Chromosomal organization of the heavy chain variable region gene segments comprising the human fetal antibody repertoire.

The adult repertoire of antibody specificities is acquired in a developmentally programmed fashion that, in mouse and man, parallels the ordered rearrangement of a limited number of germ-line heavy chain variable region (VH) gene segments during development. It has been hypothesized that this developmental bias is a consequence of gene organization. In the mouse, rearrangement of VH gene segments proximal to the heavy chain joining region (JH) locus precedes rearrangement of genes located more distal to the JH locus. Similarly, in man, two VH elements located proximal to JH are expressed during fetal development. To test further this hypothesis in man, we have determined in a single individual the positions of an additional eight distinct VH elements known to comprise a significant fraction of the human developmental repertoire. These developmentally expressed VH elements were found to be dispersed over a region of 890 kilobases of the VH locus and were interspersed with other VH elements that are not known to be developmentally expressed. Thus, the ordered developmental expression of VH gene segments in man must involve mechanisms beyond physical proximity to the JH locus. Further, these results support the notion that fetal expression of VH gene segments is a regulated process and suggest that this regulation is important in the acquisition of immunocompetence.


Functional erythroid promoters created by interaction of the transcription factor GATA-1 with CACCC and AP-1/NFE-2 elements.

We have investigated interactions between the erythroid transcription factor GATA-1 and factors binding two cis-acting elements commonly linked to GATA sites in erythroid control elements. GATA-1 is present at all stages of erythroid differentiation, is necessary for erythropoiesis, and binds sites in all erythroid control elements. However, minimal promoters containing GATA-1 sites are inactive when tested in erythroid cells. Based on this observation, two erythroid cis elements, here termed CACCC and AP-1/NFE-2, were linked to GATA sites in minimal promoters. None of the elements linked only to a TATA box created an active promoter, but GATA sites linked to either CACCC or AP-1/NFE-2 elements formed strong erythroid promoters. A mutation of T to C at position -175 in the gamma-globin promoter GATA site, associated with hereditary persistence of fetal hemoglobin (HPFH), increased expression of these promoters in both fetal and adult cells. A construct bearing the beta-globin CACCC element was more active in adult and less active in fetal erythroid cells, when compared with the gamma-globin CACCC element. These studies suggest that erythroid control elements are formed by the interactions of at least three transcription factors, none of which functions alone.


Alpha-crystallin can function as a molecular chaperone.

The alpha-crystallins (alpha A and alpha B) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, crystallins (especially alpha B) are found in many cells and organs outside the lens, and alpha B is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that alpha-crystallin can function as a molecular chaperone. Stoichiometric amounts of alpha A and alpha B suppress thermally induced aggregation of various enzymes. In particular, alpha-crystallin is very efficient in suppressing the thermally induced aggregation of beta- and gamma-crystallins, the two other major mammalian structural lens proteins. alpha-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured gamma-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that alpha-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells alpha-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.


ATPase activity of transcription-termination factor rho: functional dimer model.

Transcription-termination factor rho of Escherichia coli functions as an RNA-dependent ATPase that causes transcript release at specific rho-dependent termination sites on the DNA template. Rho exists as a hexagon of identical subunits, physically organized as a trimer of dimers with D3 symmetry. The structural asymmetry of the dimer is reflected in the binding properties of rho; each dimer has a strong and a weak binding site for both the ATP substrate and the RNA cofactor. Here we use homopolynucleotides in competition and complementation experiments to characterize the ATPase activation properties of the cofactor binding sites of the functional rho dimer. We show that (i) no ATPase activity is observed unless both the high- and the low-affinity cofactor binding sites of the functional rho dimer are occupied; (ii) saturating levels of poly(rC), poly(rC) in combination with poly(rU), or poly(rU) alone can fully activate the ATPase of rho; and (iii) poly(dC) can serve as a fully competitive inhibitor of half of the ATPase activity of rho when one of the cofactor sites is filled with poly(rC). These observations lead to a set of phenomenological rules that describe the cofactor dependence of the ATPase activation of the functional dimer of rho and help to define a mechanistic basis for interpreting rho function in termination.


CDC46/MCM5, a yeast protein whose subcellular localization is cell cycle-regulated, is involved in DNA replication at autonomously replicating sequences.

Saccharomyces cerevisiae cells containing mutations in the cell-division-cycle gene CDC46 arrest with a large bud and a single nucleus with unreplicated DNA at the non-permissive temperature. This G1/S arrest, together with the increased rates of mitotic chromosome loss and recombination phenotype, suggests that these mutants are defective in DNA replication. The subcellular localization of the CDC46 protein changes with the cell cycle; it is nuclear between the end of M phase and the G1/S transition but is cytoplasmic in other phases of the cell cycle. Here we show that CDC46 is identical to MCM5, based on complementation analysis of the mcm5-1 and cdc46-1 alleles, complementation of the minichromosome maintenance defect of mcm5-1 by CDC46, and the genetic linkage of these two genes. Like mcm5-1, cdc46-1 and cdc46-5 also show a minichromosome maintenance defect thought to be associated with DNA replication initiation at autonomously replicating sequences. Taken together, these observations suggest that CDC46/MCM5 acts during a very narrow window at the G1/S transition or the beginning of S phase by virtue of its nuclear localization to effect the initiation of DNA replication at autonomously replicating sequences.


The (4;11)(q21;q23) chromosome translocations in acute leukemias involve the VDJ recombinase.

Chromosomal region 11q23 is frequently rearranged in acute lymphocytic leukemias (ALLs) and in acute myeloid leukemias (AMLs), mostly in reciprocal exchanges with various translocation partners. The most common of these translocations is t(4;11)(q21;q23). It is present in approximately 10% of ALL patients, most frequently in very young children. We have recently cloned a region of chromosome 11, the ALL-1 locus, found to be rearranged in malignant cells from patients with the t(4;11), t(9;11), t(11;19), t(1;11), t(6;11), t(10;11), and del(11q23) chromosomal abnormalities. Here we report the cloning and characterization of chromosomal breakpoints from leukemic cells with t(4;11) aberrations. The breakpoints cluster in regions of 7-8 kilobases on both chromosomes 4 and 11. The presence of heptamer- and nonamer-like sequences at the sites of breakage suggests that the VDJ recombinase utilized for immunoglobulin gene rearrangement is also directly involved in these translocations. We also show that leukemic cells with t(4;11) express altered RNAs transcribed from the derivative chromosomes 11 and 4.


Variations in patterns of DNA damage induced in human colorectal tumor cells by 5-fluorodeoxyuridine: implications for mechanisms of resistance and cytotoxicity.

We have previously shown that treatment of the HT29 human colorectal tumor (HCT) cell line with 100 nM 5-fluorodeoxyuridine (FdUrd) induces DNA fragments ranging from 50 kilobases to 5 megabases. The studies reported here were conducted to characterize the kinetics, concentration dependence, and pharmacologic specificity of this process and to determine if such fragmentation varies among HCT cell lines. HT29 and SW620 cells yielded similar fragment size distributions upon treatment with either FdUrd or CB3717 [a folate analog inhibitor of thymidylate synthase (TS)]. With either of these agents the SW620 line required higher drug concentrations or longer incubation times than HT29 cells to achieve a given level of fragmentation or cytotoxicity, even though the two cell lines are equally sensitive to FdUrd-induced TS inhibition. These data indicate that SW620 resistance is not due to a lesion in the events leading up to TS inhibition but it may be due to a difference in the steps following TS inhibition. Aphidicolin, a DNA polymerase inhibitor, did not cause substantial fragmentation or cytotoxicity in these two cell lines, demonstrating that the fragmentation response to the other two drugs is not a general consequence of DNA synthesis inhibition. A third HCT line, HuTu80, gave rise only to a smaller and more discrete population of DNA fragments, ranging from approximately 50 to 200 kilobases, following exposure to FdUrd. Similar patterns were seen in this line upon treatment with CB3717 or aphidicolin, indicating that this fragmentation pattern is not specific to TS inhibition and may be characteristic of a more general response than that seen in the other two cell lines. DNA fragments induced by FdUrd in HuTu80 cells did not degrade into smaller pieces, demonstrating that the process by which they are formed is distinct from apoptosis. We conclude that the responses of HCT cells to FdUrd-induced TS inhibition vary significantly, that these differences may reflect heterogeneity in the mechanism of DNA damage formation, and that, in some cases, FdUrd resistance may be due to alterations in the fragmentation process.


Supercoiled DNA wraps around the bacteriophage phi 29 head-tail connector.

Supercoiled pBR322 DNA wraps around the outside of the isolated Bacillus subtilis bacteriophage phi 29 head-tail connector, the crux of the DNA packaging machine of the viral precursor capsid or prohead. The contour length of the supercoiled DNA, determined by EM, decreased by approximately 180 base pairs for each connector bound. Mass and radial density determinations by scanning transmission EM showed that the increased mass of the connector-DNA complex relative to the connector alone was equivalent to approximately 170 base pairs of DNA and was located around the outside of the connector. Topoisomerase I treatment of the complexes followed by deproteinization suggested that supercoils were restrained by the connectors. Connectors bound linear and open-circular plasmid DNAs inefficiently but were not wrapped by these DNAs. The wrapping of supercoiled DNA around the isolated phi 29 connector is hypothesized to reflect the initiation phase of the normal process of DNA packaging. Packaging substrates would be supercoiled, wrapped by the connector, linearized, and translocated by rotation of the connector relative to the viral capsid with the aid of ATP hydrolysis.


Differential regulation of neuropeptide mRNA expression in intrastriatal striatal transplants by host dopaminergic afferents.

The effects of dopamine-specific manipulations on neuropeptide gene expression in intrastriatal grafts of fetal striatal tissue were studied by quantitative in situ hybridization histochemistry, using 35S-labeled oligonucleotide probes. Messenger RNA transcripts for the striatal neuropeptides preproenkephalin (PPE) and preprotachykinin (PPT) were detected in neurons forming discrete patches in the striatal grafts. The relative abundance of PPE and PPT mRNA-expressing neurons within the graft patches (51-54%) was similar to that found in normal caudate-putamen. In specimens with intact dopamine afferents the expression of PPE mRNA in grafted neurons was similar to that found in normal caudate putamen, whereas the hybridization signal for PPT mRNA was 27% higher in the graft neurons than in the normal caudate-putamen. Removal of host dopaminergic afferents by 6-hydroxydopamine lesions of the ipsilateral mesostriatal dopamine pathway increased the hybridization signal for PPE mRNA both in the grafts (+84%) and in the spared ipsilateral host caudate-putamen (+125%), whereas the PPT signal was reduced by 53% in the grafts and by 51% in the remaining host caudate-putamen. Similarly, chronic treatment of grafted animals with the dopamine receptor antagonist haloperidol (2 mg/kg per day for 10 days) produced a 146% increase in the PPE signal in the grafts and a 175% increase in the intact contralateral caudate-putamen, whereas the signal for PPT mRNA was again decreased by 52% and 51% in the grafts and host caudate-putamen, respectively. These results show that the host nigrostriatal dopamine pathway differentially regulates enkephalin and substance P gene expression within striatal grafts and thereby exerts a tonic functional influence over grafted striatal neurons.


Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.

Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeable to Ca2+; heteromeric channels containing GluR2 subunits are Ca(2+)-impermeable. Thus, altered expression of GluR2 in development or following a neurological insult or injury to the brain can act as a switch to modify Ca2+ permeability. To investigate the molecular mechanism underlying delayed postischemic cell death, GluR1, GluR2, and GluR3 gene expression was examined by in situ hybridization in postischemic rats. Following severe, transient forebrain ischemia GluR2 gene expression was preferentially reduced in CA1 hippocampal neurons at a time point that preceded their degeneration. The switch in expression of kainate/AMPA receptor subunits coincided with the previously reported increase in Ca2+ influx into CA1 cells. Timing of the switch indicates that it may play a causal role in postischemic cell death.


Analogs of palmitoyl-CoA that are substrates for myristoyl-CoA:protein N-myristoyltransferase.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p; EC 2.3.1.97) is an essential enzyme that is highly selective for myristoyl-CoA in vivo. It is unclear why myristate (C14:0), a rare cellular fatty acid, has been selected for this covalent protein modification over more abundant fatty acids such as palmitate (C16:0), nor is it obvious how the enzyme's acyl-CoA binding site is able to discriminate between these two fatty acids. Introduction of a cis double bond between C5 and C6 of palmitate [(Z)-5-hexadecenoic acid] or a triple bond between C4 and C5 or C6 and C7 (Y4- and Y6-hexadecenoic acids) yields compounds that, when converted to their CoA derivatives, approach the activity of myristoyl-CoA as Nmt1p substrates in vitro. Kinetic studies of 42 C12-C18 fatty acids containing triple bonds, para-phenylene, or a 2,5-furyl group, as well as cis and trans double bonds, suggest that the geometry of the enzyme's acyl-CoA binding site requires that the acyl chain of active substrates assume a bent conformation in the vicinity of C5. Moreover, the distance between C1 and the bend appears to be a critical determinant for optimal positioning of the acyl-CoA in this binding site so that peptide substrates can subsequently bind in the sequential ordered bi-bi reaction mechanism. Identification of active, conformationally restricted analogs of palmitate offers an opportunity to "convert" wild-type or mutant Nmts to palmitoyltransferases so that they can deliver these C16 fatty acids to critical N-myristoylproteins in vivo. nmt181p contains a Gly-451-->Asp mutation, which causes a marked reduction in the enzyme's affinity for myristoyl-CoA. Strains of S. cerevisiae containing nmt1-181 exhibit temperature-sensitive myristic acid auxotrophy: their complete growth arrest at 37 degrees C is relieved when the medium is supplemented with 500 microM C14:0 but not with C16:0. The CoA derivatives of (Z)-5-hexadecenoic and Y6-hexadecynoic acids are as active substrates for the mutant enzyme as myristoyl-CoA at 24 degrees C. However, unlike C16:0, they produce growth arrest of nmt181p-producing cells at this "permissive" temperature, suggesting that these C16 fatty acids do not allow expression of the biological functions of essential S. cerevisiae N-myristoylproteins.


Chimeric gag-V3 virus-like particles of human immunodeficiency virus induce virus-neutralizing antibodies.

A 41-kDa unprocessed human immunodeficiency virus 2 (HIV-2) gag precursor protein that has a deletion of a portion of the viral protease assembles as virus-like particles by budding through the cytoplasmic membrane of recombinant baculovirus-infected insect cells. We have constructed six different combinations of chimeric genes by coupling the truncated HIV-2 gag gene to the neutralizing domain (V3) or the neutralizing and the CD4 binding domains (V3+CD4BD) of gp120 env gene sequences from HIV-1 or HIV-2. The env gene sequences were inserted either into the middle of the gag gene or at the 3' terminus of the gag gene. Virus-like particles were formed by chimeric gene products only when the env gene sequences were linked to the 3' terminus of the gag gene. Insertion of env gene sequence in the middle of the gag gene resulted in high-level chimeric gene expression but without the formation of virus-like particles. Three different chimeric genes [gag gene with HIV-1 V3 (1V3), gag gene with HIV-2 V3 (2V3), and gag gene with HIV-2 V3+CD4BD (2V3+CD4BD)] formed virus-like particles that were secreted into the cell culture medium. In contrast, the HIV-1 V3+CD4BD/HIV-2 gag construct did not form virus-like particles. The chimeric gag-env particles had spherical morphology and the size was slightly larger than that of the gag particles, but the chimeric particles were similar to the mature HIV particles. Western blot analysis showed that the gag-env chimeric proteins were recognized by antibodies in HIV-positive human serum and rabbit anti-gp120 serum. Rabbit anti-gag 1V3 and anti-gag 2V3 sera reacted with authentic gp120 of HIV-1 and HIV-2, respectively, and neutralized homologous HIV infectivity. Our results show that precursor gag protein has potential as a carrier for the presentation of foreign epitopes in good immunological context. The gag protein is highly immunogenic and has the ability to carry large foreign inserts; as such, it offers an attractive approach for HIV vaccine development.


Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses.

Dengue type 4 virus (DEN4) cDNA was used as a vector to express genes of the distantly related tick-borne encephalitis virus (TBEV). Full-length chimeric TBEV/DEN4 cDNAs were constructed by substituting TBEV genes coding for proteins such as capsid (C); pre-membrane, which is the precursor of membrane (M); envelope (E); or nonstructural protein NS1 for the corresponding DEN4 sequences. RNA transcripts prepared from cDNAs were used to transfect permissive simian cells. Two viable chimeric viruses that contained TBEV CME or ME genes were recovered. Compared with DEN4, chimeric TBE(ME)/DEN4 virus [designated vTBE(ME)/DEN4] produced larger plaques and grew to higher titer in simian cells. In contrast, vTBE(ME)/DEN4 produced smaller plaques on mosquito cells and grew to lower titer than DEN4. Analysis of viral RNA and proteins produced in vTBE(ME)/DEN4- and DEN4-infected mosquito or simian cells revealed that the chimera was restricted in its ability to enter and replicate in mosquito cells. In contrast, vTBE(ME)/DEN4 entered simian cells efficiently and its RNA was replicated more rapidly in these cells than was parental DEN4 RNA. Following intracerebral inoculation, vTBE(ME)/DEN4 caused fatal encephalitis in both suckling and adult mice, while nearly all mice inoculated by the same route with DEN4 did not develop disease. Unlike wild-type TBEV, vTBE(ME)/DEN4 did not cause encephalitis when adult mice were inoculated by a peripheral route. Adult mice previously inoculated with the chimera by a peripheral route were completely resistant to subsequent intraperitoneal challenge with 10(3) times the median lethal dose of TBEV, whereas mice previously inoculated with DEN4 were not protected. These findings indicate that (i) the TBEV M and E genes of the chimeric virus are major protective antigens and induce resistance to lethal TBEV challenge and (ii) other regions of the TBEV genome are essential for the ability of this virus to spread from a peripheral site to the brain. Success in constructing a viable TBEV/DEN4 chimera that retains the protective antigens of TBEV but lacks its peripheral invasiveness provides a strategy for the development of live attenuated TBEV vaccines.


A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition.

A peptide designated DP-107 was synthesized containing amino acid residues 558-595 of the envelope glycoprotein gp160 of human immunodeficiency virus type 1 strain LAI (HIV-1LAI). Algorithms for secondary structure have predicted that this region of the envelope transmembrane protein should form an extended alpha-helix. Consistent with this prediction, analysis by circular dichroism (CD) indicated that, under physiological conditions, DP-107 is approximately 85% helical. The high degree of stable secondary structure in a synthetic peptide of this size suggests self-association typical of a coiled coil or leucine zipper. In biological assays, the peptide efficiently blocked virus-mediated cell-cell fusion processes as well as infection of peripheral blood mononuclear cells by both prototypic and primary isolates of HIV-1. A single amino acid substitution in the peptide greatly destabilized its solution structure as measured by CD and abrogated its antiviral activity. An analogue containing a terminal cysteine was oxidized to form a dimer, and this modification lowered the dose required for antiviral effect from 5 to about 1 microgram/ml. These results suggest that both oligomerization and ordered structure are necessary for biological activity. They provide insights also into the role of this region in HIV infection and the potential for development of a new class of antiviral agents.