bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and characterization of a cDNA showing negative regulation in v-src-transformed 3Y1 rat fibroblasts.
Authors: Ozaki T , Sakiyama S
Published: May 2006

A differential screening procedure was used to isolate genes that are specifically down-regulated in transformed cells. After screening a cDNA library derived from normal rat fibroblast (3Y1) cells, we obtained several independent cDNAs whose mRNA level was substantially lower in 3Y1 cells transformed with Rous sarcoma virus than in untransformed 3Y1 cells. Among them, N03 cDNA has been characterized extensively. N03 mRNA was also absent from v-mos- or simian virus 40-transformed 3Y1 cells but was present in v-Ha-ras-transformed 3Y1 cells. Nucleotide sequence analysis showed that the N03 protein is composed of 178 amino acid residues and does not have any sequence similarities with proteins in the data base. A putative zinc-finger domain is located in the central part of the sequence and a proline-rich domain is in the C-terminal region. N03 mRNA was detected by Northern blot analysis in several tissues, including lung, kidney, intestine, and brain, but not in liver. Genomic Southern blot hybridization revealed that the N03 gene exists as a single copy in the rat genome and that closely related, single-copy genes are also present in chicken and human.
Pharmacologic effects in vivo in brain by vector-mediated peptide drug delivery.
Authors: Bickel U , Yoshikawa T , Landaw E M , Faull K F , Pardridge W M
Published: May 2006

Pharmacologic effects in brain caused by systemic administration of neuropeptides are prevented by poor transport of the peptide through the brain vascular endothelium, which comprises the blood-brain barrier in vivo. In the present study, successful application of a chimeric peptide approach to enhance drug delivery through the blood-brain barrier for the purpose of achieving a central nervous system pharmacologic effect is described. The chimeric peptide was formed by linkage of a potent vasoactive intestinal peptide (VIP) analogue, which had been monobiotinylated, to a drug transport vector. The vector consisted of a covalent conjugate of avidin and the OX26 monoclonal antibody to the transferrin receptor. Owing to the high concentration of transferrin receptors on brain capillary endothelia, OX26 targets brain and undergoes receptor-mediated transcytosis through the blood-brain barrier. Systemic infusion of low doses (12 micrograms/kg) of the VIP chimeric peptide in rats resulted in an in vivo central nervous system pharmacologic effect: a 65% increase in cerebral blood flow. Biotinylated VIP analogue without the brain transport vector was ineffective.
The envelope glycoprotein of human immunodeficiency virus type 1 stimulates release of neurotoxins from monocytes.
Authors: Giulian D , Wendt E , Vaca K , Noonan C A
Published: May 2006

Mononuclear phagocytes infected with human immunodeficiency virus 1 (HIV-1) produce soluble factors that kill neurons in culture. To define the molecular events that lead to neuron killing, HIV-1 proteins were tested for the ability to trigger release of neurotoxins from human monocytes and lymphocytes. None of the recombinant-derived HIV-1 proteins examined (reverse transcriptase, protease, gag, nef, or gp120) were directly neurotoxic at concentrations from 100 pM to 10 nM. The envelope glycoprotein gp120 did, however, stimulate both isolated human blood monocytes and the monocytoid line THP-1 (but not lymphocytes or the lymphoid cell line H9) to discharge neurotoxic factors. These toxins consisted of heat-stable, protease-resistant molecules (< 500 Da) that copurified with neurotoxins from HIV-1-infected THP-1 cells and were blocked by antagonists to N-methyl-D-aspartate receptors. Release of neurotoxins through gp120 stimulation involved monocytoid CD4 receptors because toxin production could be inhibited either by a monoclonal antibody to the CD4-binding region of gp120 or by soluble CD4 receptors. Alternatively, production of neuron-killing factors could be induced with a peptide from the CD4-binding region of gp120. These data show that the HIV-1 envelope glycoprotein alone can stimulate neurotoxin release by binding to CD4 receptors of mononuclear phagocytes. Such neurotoxic factors may, in turn, contribute to the central nervous system dysfunction associated with HIV-1 by acting on neurons through N-methyl-D-aspartate receptors.
Crystal structure of CATGGCCATG and its implications for A-tract bending models.
Authors: Goodsell D S , Kopka M L , Cascio D , Dickerson R E
Published: May 2006

The single-crystal x-ray analysis of orthorhombic CATGGCCATG has revealed a previously unrecognized mode of intrinsic bending in DNA. The decamer shows a smooth bend of 23 degrees over the central four base pairs, caused by preferential stacking interactions at guanine bases. The bend is produced by a roll of base pairs along their long axes, in a direction that compresses the wide major groove of the double helix. This major-groove-compressing bend at GGC, plus the abundant crystallographic evidence that runs of successive adenine bases (A-tracts) are straight and unbent, requires rethinking of the models most commonly invoked to explain A-tract bending. A decade of excellent experimental work involving gel migration experiments, cyclization kinetics, and nucleosome phasing has clearly established that introduction of short A-tracts into a general DNA sequence in synchrony with the natural repeat of the helix leads to bending. But it does not logically and inevitably follow that the actual bending is to be found within these introduced A-tracts or even at junctions with general-sequence B-DNA.
Biocatalytic synthesis of acrylates in supercritical fluids: tuning enzyme activity by changing pressure.
Authors: Kamat S V , Iwaskewycz B , Beckman E J , Russell A J
Published: May 2006

Supercritical fluids are a unique class of nonaqueous media in which biocatalytic reactions can occur. The physical properties of supercritical fluids, which include gas-like diffusivities and liquid-like densities, can be predictably controlled with changing pressure. This paper describes how adjustment of pressure, with the subsequent predictable changes of the dielectric constant and Hildebrand solubility parameter for fluoroform, ethane, sulfur hexafluoride, and propane, can be used to manipulate the activity of lipase in the transesterification of methylmethacrylate with 2-ethyl-1-hexanol. Of particular interest is that the dielectric constant of supercritical fluoroform can be tuned from approximately 1 to 8, merely by increasing pressure from 850 to 4000 psi (from 5.9 to 28 MPa). The possibility now exists to predictably alter both the selectivity and the activity of a biocatalyst merely by changing pressure.
Molecular cloning and expression of cDNA for mammalian translation initiation factor 5.
Authors: Das K , Chevesich J , Maitra U
Published: May 2006

Eukaryotic translation initiation factor 5 (eIF-5) catalyzes the hydrolysis of GTP bound to the 40S ribosomal initiation complex (40S.AUG.Met-tRNAf-eIF-2.GTP) with the subsequent joining of a 60S ribosomal subunit resulting in the formation of a functional 80S initiation complex. A rat cDNA that encodes eIF-5 has been isolated and expressed in Escherichia coli to yield a catalytically active eIF-5 protein. The 3.55-kb cDNA encodes a protein of 429 amino acids (calculated M(r) 48,926) with properties that are similar to eIF-5 isolated from rabbit reticulocyte lysates. The deduced amino acid sequence of eIF-5 contains sequence motifs characteristic of proteins of the GTPase superfamily.
Redox-activated expression of the cytosolic copper/zinc superoxide dismutase gene in Nicotiana.
Authors: Hérouart D , Van Montagu M , Inzé D
Published: May 2006

Superoxide dismutases (SODs; superoxide: superoxide oxidoreductase, EC 1.15.1.1) play a key role in protection against oxygen radicals, and SOD gene expression is highly induced during environmental stress. To determine the conditions of SOD induction, the promoter of the cytosolic copper/zinc SOD (Cu/ZnSODcyt) gene was isolated in Nicotiana plumbaginifolia and fused to the beta-glucuronidase reporter gene. Oxidative stress is likely to alter the cellular redox in favor of the oxidized status. Surprisingly, the expression of the Cu/ZnSODcyt gene is induced by sulfhydryl antioxidants such as reduced glutathione, cysteine, and dithiothreitol, whereas the oxidized forms of glutathione and cysteine have no effect. It is therefore possible that reduced glutathione directly acts as an antioxidant and simultaneously activates the Cu/ZnSODcyt gene during oxidative stress.
Nuclear magnetic resonance imaging and spectroscopy of human brain function.
Authors: Shulman R G , Blamire A M , Rothman D L , McCarthy G
Published: May 2006

The techniques of in vivo magnetic resonance (MR) imaging and spectroscopy have been established over the past two decades. Recent applications of these methods to study human brain function have become a rapidly growing area of research. The development of methods using standard MR contrast agents within the cerebral vasculature has allowed measurements of regional cerebral blood volume (rCBV), which are activity dependent. Subsequent investigations linked the MR relaxation properties of brain tissue to blood oxygenation levels which are also modulated by consumption and blood flow (rCBF). These methods have allowed mapping of brain activity in human visual and motor cortex as well as in areas of the frontal lobe involved in language. The methods have high enough spatial and temporal sensitivity to be used in individual subjects. MR spectroscopy of proton and carbon-13 nuclei has been used to measure rates of glucose transport and metabolism in the human brain. The steady-state measurements of brain glucose concentrations can be used to monitor the glycolytic flux, whereas subsequent glucose metabolism--i.e., the flux into the cerebral glutamate pool--can be used to measure tricarboxylic acid cycle flux. Under visual stimulation the concentration of lactate in the visual cortex has been shown to increase by MR spectroscopy. This increase is compatible with an increase of anaerobic glycolysis under these conditions as earlier proposed from positron emission tomography studies. It is shown how MR spectroscopy can extend this understanding of brain metabolism.
Separation between cytosolic calcium and secretion in chromaffin cells superfused with calcium ramps.
Authors: Michelena P , García-Pérez L E , Artalejo A R , García A G
Published: May 2006

This paper describes experiments in which cytosolic Ca2+ concentrations ([Ca2+]i) and catecholamine release were measured in two populations of chromaffin cells stimulated with a solution enriched in K+ (100 mM). Once depolarized, external Ca2+ or Ba2+ ions were offered to cells either as a single 2.5 mM step or as a ramp that linearly increased the concentration from 0 to 2.5 mM over a 10-min period. A clear separation between the changes of the [Ca2+]i and the time course of secretion was observed. Specifically, secretion and [Ca2+]i rose in parallel when a Ca2+ step was used to reach a peak in a few seconds; however, while secretion declined to the basal level, [Ca2+]i remained elevated at a plateau of 400 nM. With a Ca2+ ramp, only a transient small peak of secretion was observed, yet the [Ca2+]i remained elevated throughout the 10-min stimulation period. The separation between secretion and [Ca2+]i was observed even when voltage-dependent Ca2+ channels were expected to remain open (mild depolarization in the presence of 1 microM Bay K 8644). By using Ba2+ steps or ramps, sustained noninactivating secretory responses were obtained. The results suggest that the rate and extent of secretion are not a simple function of the [Ca2+]i at a given time; they are compatible with the following conclusions: (i) A steep extracellular-to-cytosolic Ca2+ gradient is required to produce a sharp increase in the [Ca2+]i at exocytotic sites capable of evoking a fast but transient secretory response. (ii) As a result of Cai(2+)-dependent inactivation of Ca2+ channels, those high [Ca2+]i are possible only at early times after cell depolarization. (iii) The Cai(2+)-dependent supply of storage granules to the secretory machinery cooperates with the supply of Ca2+ through Ca2+ channels to regulate the rate and extent of secretion.
Regulated expression of an insulin-responsive glucose transporter (GLUT4) minigene in 3T3-L1 adipocytes and transgenic mice.
Authors: Ezaki O , Flores-Riveros J R , Kaestner K H , Gearhart J , Lane M D
Published: May 2006

Preliminary studies showed that up to 7 kb of 5' flanking sequence of the insulin-responsive glucose transporter (GLUT4) gene are insufficient to mediate differentiation-induced reporter gene expression in mouse 3T3-L1 preadipocytes. To locate the regulatory element(s) responsible for this function, a minigene containing the entire GLUT4 gene with substantial 5' and 3' flanking sequence and a short segment of foreign DNA (for transcript identification) was constructed and transfected into mice and 3T3-L1 preadipocytes at relatively low copy number. In transgenic mice the GLUT4 minigene exhibited a pattern of tissue-specific expression similar, but not identical, to that of the endogenous gene. In 3T3-L1 cells expression of minigene mRNA occurred upon differentiation into adipocytes, with kinetics virtually identical to that of endogenous GLUT4 mRNA. In both cultured adipocytes and transgenic mice, the level of expression of the minigene was low relative to that of the endogenous gene. Treatment of minigene-transfected 3T3-L1 adipocytes with 8-bromo-cAMP, which represses transcription of the endogenous GLUT4 gene, also repressed expression of the GLUT4 minigene. However, insulin, which down-regulates transcription of the endogenous GLUT4 gene, failed to normally down-regulate expression of the GLUT4 minigene. These findings indicate that the cis-acting elements required for directing tissue-specific expression (in heart, skeletal muscle, and brown adipose tissue), differentiation-induced activation of transcription, and cAMP-induced repression of transcription are located within the 14-kb GLUT4 minigene. However, the cis elements necessary for maximal tissue-specific expression and for insulin-induced down-regulation of expression are not located in the minigene.
Reactive oxygen species and neutrophil respiratory burst cytochrome b558 are produced by kidney glomerular cells in passive Heymann nephritis.
Authors: Neale T J , Ullrich R , Ojha P , Poczewski H , Verhoeven A J , Kerjaschki D
Published: May 2006

Reactive oxygen species (ROS) have been implicated in the production of glomerular damage in passive Heymann nephritis (PHN), an experimental form of membranous nephropathy with neutrophil-independent proteinuria. Immunohistochemistry with monoclonal antibodies specific for cytochrome b558 (a major component of the oxidoreductase complex of the respiratory burst in stimulated neutrophilic granulocytes) showed that this enzyme is localized within visceral glomerular epithelial cells (GECs) in a dense, granular pattern in rats with PHN and proteinuria. By immunoelectron-microscopy, the cytochrome was found in membrane vesicles within the GEC and also extracellularly on the GEC membranes facing the glomerular basement membrane (GBM). By immunoblotting, cytochrome b558 was detected in highest concentration in lysates of isolated glomeruli from proteinuric rats. By contrast, only traces were found in normal glomeruli by immunohistochemistry. Depletion of complement abolished the expression of the cytochrome. Using an ultrastructural cerium-H2O2 histochemistry technique, the functional activity of the glomerular ROS-generating system was demonstrated exclusively in proteinuric PHN, where H2O2 was found in highest concentration within the GBM. These results provide evidence that in rats with PHN and proteinuria, the GECs express and externalize respiratory-burst enzymes that generate ROS in a manner similar to neutrophilic granulocytes, which could then lead to glomerular damage.
Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.
Authors: Liggett S B , Freedman N J , Schwinn D A , Lefkowitz R J
Published: May 2006

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.
Interleukin 12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist.
Authors: Tripp C S , Wolf S F , Unanue E R
Published: May 2006

Listeriosis in mice with the severe combined immunodeficiency (SCID) mutation is an established model in vivo and in vitro of interferon gamma (IFN-gamma)-dependent macrophage activation by natural killer (NK) cells during the development of natural immunity. We demonstrate that IFN-gamma production from SCID splenocytes is stimulated by interleukin (IL) 12, tumor necrosis factor alpha (TNF-alpha), and IL-2 but is inhibited by IL-10, IL-10, IL-12, and TNF are induced by heat-killed Listeria monocytogenes (hk-LM) from SCID splenocytes and peritoneal macrophages. IL-12 production is necessary for hk-LM to stimulate IFN-gamma production by SCID splenocytes since neutralization of IL-12 totally blocks IFN-gamma production in this system. TNF-alpha and IL-2 act synergistically with IL-12 to augment IFN-gamma production. Also, exogenous IL-2 increases the response of NK cells to hk-LM or to IL-12 and TNF-alpha. In contrast, IL-10 inhibits hk-LM-induced IFN-gamma production at two levels: (i) by inhibiting TNF and IL-12 production from these cultures (presumably from the macrophage) and (ii) by inhibiting the stimulatory effects of IL-12 and TNF-alpha on NK-cell IFN-gamma production. Thus, these data indicate that macrophage production of TNF-alpha and IL-12 stimulates the release of IFN-gamma by NK cells and that IL-10 produced in response to hk-LM inhibits this response at the level of the macrophage and the NK cell.
Purification of a galanin receptor from pig brain.
Authors: Chen Y , Fournier A , Couvineau A , Laburthe M , Amiranoff B
Published: May 2006

A galanin receptor protein was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) from pig brain membranes and then purified by single-step affinity chromatography. The product exhibits saturable and specific binding for galanin with a binding activity of 17 nmol/mg of protein and a dissociation constant (Kd) of 10 nM. This represents a 300,000-fold purification over the detergent-solubilized fraction with a final recovery of 31% of the initial membrane galanin binding activity. Gel electrophoresis of the affinity-purified material showed a single polypeptide of 54 kDa by silver staining and after radioiodination. Cross-linking of a purified fraction affinity-labeled with 125I-labeled galanin revealed a single band for the galanin-receptor complex at 57 kDa. The general binding characteristics of the purified preparation appeared to be identical to those of the crude soluble material as far as specificity toward galanin and the structural requirement for galanin are concerned. In contrast, unlike the CHAPS-soluble galanin receptor, binding of 125I-labeled galanin to the purified galanin receptor was not sensitive to guanine nucleotides, suggesting that dissociation of the inhibitory guanine nucleotide binding protein from the galanin receptor occurred during purification. The purification to homogeneity of a galanin receptor paves the way toward its sequencing and cloning.
Retroviral insertions into a herpesvirus are clustered at the junctions of the short repeat and short unique sequences.
Authors: Jones D , Isfort R , Witter R , Kost R , Kung H J
Published: May 2006

We previously described the integration of a nonacute retrovirus, reticuloendotheliosis virus (REV), into the genome of a herpesvirus, Marek disease virus (MDV), following both long-term and short-term coinfection in cultured fibroblasts. The long-term coinfection occurred in the course of attenuating the oncogenicity of the JM strain of MDV and was sustained for > 100 passages. The short-term coinfection, which spanned only 16 passages, was designed to recreate the insertion phenomenon under controlled conditions. We found that REV integrations into MDV were common and could occur within the first passage following coinfection. Now we have mapped the integration sites. After 5-16 passages in vitro, 17 out of 19 REV junction sites are clustered in two 1-kilobase regions at the junctions of the short unique and short repeat region of MDV. In the long-term cocultivation experiment, 6 out of 10 insertions also mapped in this region. In both cases, integrated proviruses are unstable and undergo subsequent recombinative deletion, often leaving a solitary long terminal repeat. The long terminal repeat sequences are, however, stably maintained for many rounds of passaging in vitro. This clustering of insertions presumably is influenced by selection for viable and fast-growing viruses, and occurs in a region of the MDV genome which shows significant size heterogeneity in several strains.
Glucose modulates the binding of an islet-specific factor to a conserved sequence within the rat I and the human insulin promoters.
Authors: Melloul D , Ben-Neriah Y , Cerasi E
Published: May 2006

In cultured rat and human pancreatic islets, glucose stimulated transcription of the rat insulin I gene through the mini-enhancer (FF) located between residues -196 and -247. The glucose-sensitive element was delineated to the region -193 to -227. The mini-enhancer bound islet nuclear proteins to form three major complexes (C1-C3). A 22-bp subfragment, spanning the sequence -206 to -227, was sufficient to retain all binding activities of the entire FF. The homologous sequence of the human insulin promoter interacted with rat islet nuclear extracts to form a single complex, corresponding to the C1 complex of the rat insulin I sequence. C1 was present only in insulin-producing cells; it was the major complex detected in isolated human islets with both rat and human insulin sequences. Furthermore, the DNA binding activity of the C1 factor(s) was selectively modulated by extracellular glucose in a dose-dependent manner; a 4.5-fold increase in binding intensity was detected when rat islets were incubated for 1-3 h in the presence of 20 vs. 1-2 mM glucose. We therefore suggest that the factor(s) involved in the C1 complex corresponds to the glucose-sensitive factor and, consequently, may play a determining role in glucose-regulated expression of the insulin gene.
In vivo expression of mutant preproendothelins: hierarchy of processing events but no strict requirement of Trp-Val at the processing site.
Authors: Fabbrini M S , Vitale A , Pedrazzini E , Nitti G , Zamai M , Tamburin M , Caiolfa V R , Patrono C , Benatti L
Published: May 2006

Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide, originates in human cells from a 212-amino acid precursor (preproET-1). Big ET-1, an intermediate form of 38 amino acids, is generated by cleavage at basic-pair residues of proET-1, while a specific "ET-converting enzyme" was proposed to process the unusual Trp-Val site at positions 21 and 22 of big ET-1. We have previously shown that expression of synthetic RNA encoding human preproET-1 in Xenopus oocytes results in secretion of putative ET-1 and big ET-1. Here, to further dissect the processing pathway of preproET-1, we designed and expressed in oocytes a set of preproET-1 mutants. Four mutants affecting the Trp-Val site always originated putative ET-1(s) at levels comparable to the wild type, suggesting that there is only a conformational requirement for cleavage at this site. An Arg-->Ile mutation at the basic-pair site after the C terminus of big ET-1 fully inhibited the formation of both big ET-1 and ET-1, indicating that processing at this site is an early event and that big ET-1 is an obligate intermediate for the synthesis of ET-1 in vivo. Also, a truncated mutant bearing a stop codon after the C terminus of the big ET-1 sequence was totally stable and further processed into mature big ET-1 and ET-1, indicating that the second part of the precursor is not necessary for maturation.
Gene inoculation generates immune responses against human immunodeficiency virus type 1.
Authors: Wang B , Ugen K E , Srikantan V , Agadjanyan M G , Dang K , Refaeli Y , Sato A I , Boyer J , Williams W V , Weiner D B
Published: May 2006

Recently, immunization techniques in which DNA constructs are introduced directly into mammalian tissue in vivo have been developed. In theory, gene inoculation should result in the production of antigenic proteins in a natural form in the immunized host. Here we present the use of such a technique for the inoculation of mice with a human immunodeficiency virus type 1 (HIV-1) envelope DNA construct (pM160). Mice were injected intramuscularly with pM160 and were subsequently analyzed for their anti-HIV envelope immune responses. Antisera collected from inoculated animals reacted with the recombinant HIV-1 envelope in ELISA and immunoprecipitation assays. The antisera also contained antibodies that were able to neutralize HIV-1 infection and inhibit HIV-1-mediated syncytium formation in vitro. Furthermore, splenic lymphocytes derived from pM160-inoculated animals demonstrated HIV-envelope-specific proliferative responses. The gene inoculation technique mimics features of vaccination with live attenuated viruses and, therefore, may ultimately prove useful in the rapid development of safe and efficacious vaccines as it provides for production of relevant antigen in vivo without the use of infectious agents.
Sequence-specific scission of DNA by the chemical nuclease activity of 1,10-phenanthroline-copper(I) targeted by RNA.
Authors: Chen C B , Gorin M B , Sigman D S
Published: May 2006

RNAs modified with the chemical nuclease 1,10-phenanthroline-copper(I) can achieve the sequence-specific scission of single- and double-stranded DNA targets. The RNAs are prepared in vitro by using 5-(3-aminoallyl)-UTP as the sole source of UTP and can be readily modified with 1,10-phenanthroline by using N-succinimidyl 3-(2-pyridyl-dithio)propionate (SPDP) to cross-link the ligand to the aminoallyl moiety. Single-stranded DNAs are efficiently cleaved at multiple sites because 1,10-phenanthroline is incorporated at several uridines in the sequence. Sequence-specific double-stranded scission of duplex DNA can also be accomplished with 1,10-phenanthroline-derivatized RNA within R loops. These triple-stranded structures form in 70% formamide and involve the displacement of one strand of DNA by the RNA of identical sequence. R loop-directed scission is the first method for DNA scission applicable to any sequence. A unique application of R loop-targeted nucleolytic scission, which relies on its ability to cut DNA at any sequence, is the determination of the distance between two marker DNA sequences within a target. In this case, 1,10-phenanthroline-linked RNAs are prepared from the two distinct sequences and used to cut the DNA fragment after R-loop formation. The size of the fragment liberated by these methods is a direct measure in base pairs of the distance between the two DNA sequences. For example, the distance separating two chicken delta crystallin (delta 1 and delta 2) genes has been confirmed as 24 kilobases by this method.
DNA sequencing: modular primers assembled from a library of hexamers or pentamers.
Authors: Kotler L E , Zevin-Sonkin D , Sobolev I A , Beskin A D , Ulanovsky L E
Published: May 2006

Here we report a striking effect displayed by "modular primers," which consist of hexamer or pentamer oligonucleotide modules base-stacked to each other upon annealing to a DNA template. Such a combination of modules is found to prime DNA sequencing reactions uniquely, unlike either of the modules alone. We attribute this effect in part to the increase in the affinity of an oligonucleotide for the template in the presence of an adjacent module. All possible pentamer (or hexamer) sequences total 1024 (or 4096) samples, a manageable size for a presynthesized library. This approach can replace the synthesis of primers, which is the current bottleneck in time and cost of the primer walking sequencing, and can allow full automation of the closed cycle of walking.
Three-dimensional structure of a human immunoglobulin with a hinge deletion.
Authors: Guddat L W , Herron J N , Edmundson A B
Published: May 2006

X-ray analysis at 3.2-A resolution revealed that the Mcg IgG1 (lambda chain) immunoglobulin is a compact T-shaped molecule. Because of the hinge deletion, the Fc fragment lobe is pulled tightly upward into the junction of the Fab arms. Along the molecular twofold axis, the Fab arms are joined by an interchain disulfide bond between the two light chains. The antigen combining sites consist of large irregular cavities at the tips of the Fab regions. Potential complement (C1q) binding sites on Fc are sterically shielded by the Fab arms, but putative attachment sites are accessible for docking with the FcRI receptor on human monocytes and with protein A of Staphylococcus aureus.
Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis.
Authors: Ellis-Davies G C , Kaplan J H
Published: May 2006

The synthesis and properties of a caged calcium are described. The compound is an ortho-nitrophenyl derivative of EGTA. It is synthesized in 10 steps and with 24% overall yield. The photosensitive chelator, nitrophenyl-EGTA, has a Kd value for Ca2+ of 80 nM and for Mg2+ of 9 mM. Upon exposure to UV radiation (approximately 350 nm), the chelator is cleaved, yielding iminodiacetic acid photoproducts with low Ca affinity (Kd = 1 mM). The quantum yield of photolysis of nitrophenyl-EGTA in the presence of Ca2+ is 0.23 and in the absence of Ca2+ is 0.20. In experiments with chemically skinned skeletal muscle fibers, a fully relaxed fiber equilibrated with nitrophenyl-EGTA-Ca2+ complex, in the presence of 1 mM free Mg2+, maximally contracted after a single flash from a frequency-doubled ruby laser (347 nm). Half-maximal tension was achieved in 18 ms at 15 degrees C. Nitrophenyl-EGTA provides a tool for the investigation of the mechanism of Ca(2+)-dependent physiological processes, since under conditions of normal intracellular Ca2+ and Mg2+ concentrations, only Ca2+ is bound by the photolabile chelator and on illumination released rapidly and in high photochemical yield.
Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange.
Authors: Maret W
Published: May 2006

Mammalian metallothionein has been postulated to play a pivotal role in cellular zinc distribution. All seven of its metal atoms are bound with high thermodynamic stability in two clusters buried deeply in the molecule. If the protein is to function in metal delivery, there must be a biological mechanism to facilitate metal release. One means to achieve this would be a labilization of the clusters by interaction of metallothionein with an appropriate cellular ligand. To search for such a mediator, we have designed a rapid radiochromatographic method that can detect changes in the zinc content of 65Zn-labeled metallothionein in response to other biomolecules. Using this methodology, we have established that rabbit liver metallothionein 2 interacts with glutathione disulfide with concomitant release of zinc. Under conditions of pseudo-first-order kinetics, the monophasic reaction depends linearly on the concentration of glutathione disulfide in the range from 5 to 30 mM with a second-order rate constant k = 4.9 x 10(-3)s-1.M-1 (pH 8.6; 25 degrees C). Apparently, zinc release does not involve direct access of glutathione disulfide to the inner coordination sphere of the metals. Rather it appears that the solvent-accessible zinc-bound thiolates in two clefts of each domain of metallothionein [Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xuong, N. H., Furey, W. F., Wang, B. C. & Stout, C. D. (1991) J. Mol. Biol. 221, 1269-1293] participate in a thiol/disulfide interchange with glutathione disulfide. This rate-limiting initial S-thiolation, which occurs with indistinguishable rates in both clusters, then causes the clusters to collapse and release their zinc. Such a mechanism of metal release would link the control of the metal content of metallothionein to the cellular glutathione redox status and raises important questions about the physiological implications of this observation with regard to a role of glutathione in zinc metabolism and in making zinc available for other biomolecules.
Histidine-367 of the human common beta chain of the receptor is critical for high-affinity binding of human granulocyte-macrophage colony-stimulating factor.
Authors: Lock P , Metcalf D , Nicola N A
Published: May 2006

High-affinity receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3, and interleukin 5 consist of ligand-specific alpha chains (low-affinity subunits) and a common beta chain (beta c) that converts each complex to a high-affinity form. Although beta c alone has no detectable cytokine-binding activity, amino acid substitutions for Glu-21 of human GM-CSF significantly reduce high-affinity but not low-affinity binding, implying that beta c interacts directly with GM-CSF during formation of the high-affinity receptor but only in the presence of the alpha chain. A potential GM-CSF-binding determinant was identified in the second hemopoietin domain of beta c, and the role of individual residues within this region was investigated by determining the ability of mutated beta c chains to confer high-affinity binding when coexpressed with the alpha subunit of the GM-CSF receptor in COS cells. Substitutions involving Met-363, Arg-364, Tyr-365, and Glu-366 did not affect high-affinity binding. However, substitution of His-367 by lysine or glutamine abolished high-affinity binding, suggesting that this residue may form an important part of the high-affinity GM-CSF-binding determinant. Consistent with the loss of high-affinity binding, higher concentrations of human GM-CSF were required to stimulate proliferation of CTLL-2 cell lines transfected with cDNAs for GM-CSF receptor alpha chain and His-367 beta c mutant than those expressing GM-CSF receptor alpha subunit and beta c wild type.
Delineation of the subunit composition of human proteasomes using antisera against the major histocompatibility complex-encoded LMP2 and LMP7 subunits.
Authors: Patel S D , Monaco J J , McDevitt H O
Published: May 2006

The products of the Lmp2 and Lmp7 genes located in the major histocompatibility complex (MHC) class II region are postulated to form part of the proteasome complex. This large, multisubunit complex forms the major, nonlysosomal proteolytic machinery for the degradation of endogenous proteins and has been suggested to play a role in the processing of antigens presented by MHC class I molecules. The role of the MHC-encoded subunits within the proteasome has however remained enigmatic. To study this role, we have raised antibodies to recombinant LMP2 and LMP7 proteins. Under denaturing conditions, the anti-LMP7 serum recognizes one subunit of proteasome, whereas the anti-LMP2 serum recognizes two subunits, which may represent different forms of LMP2. The specificity of these sera has been ascertained by a lack of reactivity in T2 cells, which lack both genes. Furthermore under native conditions the anti-LMP2 serum immunoprecipitates a complex that is similar to proteasome but lacks several subunits, including LMP7. Preclearing experiments using this serum and a monoclonal antibody (2-17) specific for the non-MHC-encoded C2 proteasome subunit demonstrate that the complexes recognized by these two sera are distinct and that four subunits are unique to the complex precipitated by the anti-LMP2 serum. Interestingly, the different forms of LMP2 are segregated between these complexes. The relationship of the two complexes is discussed.
Recombinant human Fab to glycoprotein D neutralizes infectivity and prevents cell-to-cell transmission of herpes simplex viruses 1 and 2 in vitro.
Authors: Burioni R , Williamson R A , Sanna P P , Bloom F E , Burton D R
Published: May 2006

Herpes simplex viruses 1 and 2 (HSV-1 and -2) are associated with a number of conditions of varying severity, which are only partially responsive to current therapies. Human antibodies to the viruses offer a potential alternative. We describe here the generation of panels of human monoclonal Fab fragments to HSV-1 and -2 by panning a phage display combinatorial antibody library against whole lysates from the two viruses. Each lysate selected a largely distinct set of Fabs, although all of the Fabs were cross-reactive with both viruses. In a plaque-reduction assay, one Fab neutralized HSV-1 at 0.25 microgram/ml (50% reduction) and HSV-2 at 0.05 microgram/ml. This Fab also inhibited plaque formation when applied to virus-infected monolayers, completely abolishing HSV-2 plaque development at 25 micrograms/ml 72 hr postinfection, indicating the ability of the Fab to prevent cell-to-cell spread of virus. The Fab was shown to recognize viral glycoprotein D and to neutralize virus primarily by a postattachment mechanism. Recombinant Fabs may be useful for topical administration, although whole antibody will probably be required for systemic use.
Kappa B enhancer-binding complexes that do not contain NF-kappa B are developmentally regulated in mammalian brain.
Authors: Cauley K , Verma I M
Published: May 2006

DNA-binding complexes which are temporally regulated in developing rat brain have been identified by their ability to interact with the kappa B enhancer sequence in electrophoretic mobility-shift assays. These complexes, referred to as developing-brain factors (DBFs) 1 and 2, are abundant in nuclear extracts from developing rat brain through postnatal day 2 and decline to nearly undetectable levels by postnatal day 7. DBFs were not detected in extracts from cultured cell lines or in tissues other than the developing brain. The highest level of DBF DNA-binding activity was observed in developing cortex, and the lowest in cerebellum. In UV crosslinking experiments, a labeled kappa B oligonucleotide probe crosslinked 110- and 115-kDa proteins from DBF complexes. DBFs are likely to be involved in the regulation of transcriptional events which take place during brain development.
Auxotrophs of Plasmodium falciparum dependent on p-aminobenzoic acid for growth.
Authors: McConkey G A , Ittarat I , Meshnick S R , McCutchan T F
Published: May 2006

The isolation of auxotrophic strains of a parasite offers new opportunities for studying parasitology. We have isolated cloned lines of Plasmodium falciparum that, unlike the parent line from which they were derived, rely on exogenous p-aminobenzoic acid (PABA) for growth. Isolation involved random mutagenesis of a cloned line of P. falciparum and subsequent selection of PABA-dependent parasites. Both parent and PABA-dependent clones were analyzed for PABA uptake and synthesis. Each clone takes up comparable amounts of PABA from the medium. The parent line, clone 3D7, can synthesize PABA de novo, whereas the PABA-dependent clones cannot. The requirement of exogenous PABA for growth by the auxotrophic strains coupled with their inability to synthesize PABA indicates that normal parasite growth can be completely supported by either synthesis or salvage. This work further clarifies the relationship between the availability of PABA and success of the parasite, an issue of debate from classic studies showing reduced parasite load in individuals on milk-fed diets.
Mutational analysis of yeast mRNA capping enzyme.
Authors: Schwer B , Shuman S
Published: May 2006

RNA guanylyltransferase (capping enzyme) catalyzes the transfer of GMP from GTP to the 5'-diphosphate end of mRNA. The capping reaction proceeds via an enzyme-guanylate intermediate in which GMP is linked covalently to a lysine residue of the enzyme. In the capping enzyme of Saccharomyces cerevisiae, GMP is attached to a 52-kDa polypeptide, identified as the product of the essential CEG1 gene. The amino acid sequence of the CEG1 protein includes a motif, Lys70-Thr-Asp-Gly, that is conserved at the active site of vaccinia virus RNA guanylyltransferase and which is similar to the KXDG sequence found at the active sites of RNA and DNA ligases. To evaluate the role of this motif in the function of the yeast enzyme, we have expressed the CEG1 protein in active form in Escherichia coli. Replacement of Lys70 or Gly73 with alanine abrogated enzyme-guanylate formation in vitro; in contrast, alanine substitutions at Thr71 or Asp72 merely reduced activity relative to wild-type enzyme. The K70A and G73A mutations were lethal to yeast, whereas yeast carrying the T71A and D72A alleles of CEG1 were viable. These results implicate Lys70 as the active site of yeast guanylyltransferase and provide evidence that cap formation per se is an essential function in eukaryotic cells.
The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression.
Authors: Witzgall R , O'Leary E , Leaf A , Onaldi D , Bonventre J V
Published: May 2006

We have previously reported the cloning, sequencing, and partial characterization of Kid-1, a zinc finger-encoding cDNA from the rat kidney. The Kid-1 protein and approximately one-third of all other zinc finger proteins contain a highly conserved region of approximately 75 amino acids at their NH2 terminus named Krüppel-associated box (KRAB), which is subdivided into A and B domains. The evolutionary conservation, wide distribution, and genomic organization of the KRAB domains suggest an important role of this region in the transcriptional regulatory function of zinc finger proteins. The functional significance of the KRAB domain was evaluated by studying transcriptional activities of yeast GAL4-rat Kid-1 fusion proteins containing various regions of the non-zinc-finger domain of Kid-1. Transcriptional repressor activity of GAL4-Kid-1 fusion proteins maps to the KRAB-A domain. The KRAB-A domain of another zinc finger protein, ZNF2, also has repressor activity. Site-directed mutagenesis of conserved amino acids in this motif results in decreased repressor activity. Thus, we have established a functional significance for the KRAB-A domain, a consensus sequence common in zinc finger proteins.
Electroporation-mediated replacement of a positively and negatively selectable beta-tubulin gene in Tetrahymena thermophila.
Authors: Gaertig J , Thatcher T H , Gu L , Gorovsky M A
Published: May 2006

Replacement of lysine-350 by methionine in the beta-tubulin gene of Chlamydomonas confers resistance to microtubule-depolymerizing drugs and increased sensitivity to the microtubule-stabilizing drug taxol. This mutation was created in cloned BTU1, one of two coexpressed beta-tublin genes of Tetrahymena thermophila. When introduced by electroporation, the mutated gene transformed Tetrahymena exclusively by gene replacement at the homologous locus. Taxol-sensitive transformants could be retransformed with a wild-type gene and selection for taxol resistance. Analyses of phenotypic assortment and of the mRNA in transformed cells suggest that complete replacement of the BTU1 gene in the polyploid macronucleus can be obtained. These studies demonstrate the utility of this marker for studying tublin gene function and show that electroporation allows facile gene replacement in Tetrahymena.
Rapid and accurate estimates of statistical significance for sequence data base searches.
Authors: Waterman M S , Vingron M
Published: May 2006

A central question in sequence comparison is the statistical significance of an observed similarity. For local alignment containing gaps to optimize sequence similarity this problem has so far not been solved mathematically. Using as a basis the Chen-Stein theory of Poisson approximation, we present a practical method to approximate the probability that a local alignment score is a result of chance alone. For a set of similarity scores and gap penalties only one simulation of random alignments needs to be calculated to derive the key information allowing us to estimate the significance of any alignment calculated under this setting. We present applications to data base searching and the analysis of pairwise and self-comparisons of proteins.
Cellular transformation by a transmembrane peptide: structural requirements for the bovine papillomavirus E5 oncoprotein.
Authors: Meyer A N , Xu Y F , Webster M K , Smith A E , Donoghue D J
Published: May 2006

The E5 oncoprotein of bovine papillomavirus, only 44 amino acids long, occurs as a disulfide-bonded transmembrane dimer. This remarkable oncoprotein stimulates signal transduction through activation of the platelet-derived growth factor (PDGF) receptor, and E5 exhibits limited amino acid sequence similarity with PDGF. Results presented here suggest that a key feature of the hydrophobic transmembrane domain is an amino acid side chain that participates in interhelical hydrogen bond formation. These data are reminiscent of the activated neu oncogene, in which a point mutation in the transmembrane domain leads to ligand-independent dimerization and activation of a receptor tyrosine kinase. Significantly, the transmembrane domain of E5 can be largely replaced by the transmembrane domain from the activated neu receptor tyrosine kinase. Extensive mutagenesis defines the minimal structural features required for transformation by the E5 oncoprotein as, first, the ability to dimerize and, second, presentation of a negatively charged residue at the extracellular side of the membrane. The biological activity of E5 mutants that lack most amino acid residues similar to PDGF suggests that E5 and PDGF activate the PDGF receptor by distinct mechanisms.