bluuebunny/crossref_metadata_2025 huggingface dataset:
Region of peptide 125-147 of acetylcholine receptor alpha subunit is exposed at neuromuscular junction and induces experimental autoimmune myasthenia gravis, T-cell immunity, and modulating autoantibodies.
Authors: Lennon V A , McCormick D J , Lambert E H , Griesmann G E , Atassi M Z
Published: May 2006

A major antigenic region of native nicotinic acetylcholine receptors (AcChoR) has been identified by using a synthetic disulfide-looped peptide corresponding to alpha-subunit residues 125-147 of Torpedo electric organ AcChoR: Lys-Ser-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe- Pro-Phe-Asp-Gln-Gln-Asn-Cys-Thr-Met-Lys-Leu-Gly. The peptide bound 26-56% of polyclonal antibodies induced in rat, rabbit, and dog by immunization with native AcChoR. Rats inoculated with 50 micrograms of unconjugated peptide developed helper T-cell responses, delayed hypersensitivity, and antibodies to native AcChoR. Anti-peptide antibodies were more reactive with native than denatured AcChoR and bound to the alpha subunit. Some reacted exclusively with mammalian muscle AcChoR, some induced modulation of AcChoR on cultured myotubes, but none inhibited binding of alpha-bungarotoxin to solubilized or membrane-associated AcChoR. Repeated immunization induced experimental autoimmune myasthenia gravis: clinical signs in one rat and electrophysiologic and/or biochemical signs in 10 of 11 rats. Thus, at least part of the corresponding region of the mammalian AcChoR alpha subunit is extracellular at the neuromuscular junction and a potential target for pathogenic autoantibodies in patients with acquired myasthenia gravis.
Retrovirus-induced spongiform encephalopathy: the 3'-end long terminal repeat-containing viral sequences influence the incidence of the disease and the specificity of the neurological syndrome.
Authors: DesGroseillers L , Rassart E , Robitaille Y , Jolicoeur P
Published: May 2006

Using chimeric murine leukemia viruses (MuLVs) constructed in vitro with parental viral genomes from the neurotropic Cas-BR-E MuLV and the nonneurotropic amphotropic 4070-A MuLV, we previously mapped the paralysis-inducing determinant of Cas-BR-E MuLV within a pol-env region. To assess the role of the long terminal repeats (LTRs) in influencing the neurological disease, we constructed another chimeric MuLV (pNEMO-1)m harboring the gag-pol-env from Cas-BR-E MuLV and the LTR region from the strongly T-cell tropic Moloney MuLV. Although the Cas-BR-E MuLV induced mainly nonthymic leukemia, pNEMO-1 MuLV induced a thymic form of leukemia, as the parental Moloney MuLV. The pNEMO-1 MuLV induced neurological diseases less frequently than Cas-BR-E MuLV when inoculated intraperitoneally into NIH/Swiss, SIM.S, and SWR/J mice. However, it induced neurological disorders more frequently and with a shorter latency than Cas-BR-E MuLV when inoculated intrathymically. Most mice with a neurological disorder induced with pNEMO-1 MuLV showed a new clinical syndrome not usually seen with the parental Cas-BR-E MuLV: They had no lower limb paralysis but were excessively tremulous, spastic, and immobile. The topographical distribution of the spongiform degeneration in the brain of mice with this new syndrome was different from that seen in mice with lower limb paralysis induced by Cas-BR-E MuLV. These results indicate that the 1.0-kilobase-pair Cla I-Pvu I LTR-containing fragment harbors sequences influencing the incidence and the clinical manifestation of the neurological disease and suggest a specificity of LTR sequences for a new tissue (brain).
Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase.
Authors: Inoue T , Cech T R
Published: May 2006

The structure of the intervening sequence (IVS) of the Tetrahymena rRNA precursor mediates cleavage-ligation reactions that result in pre-rRNA splicing and IVS cyclization. We have developed a method for RNA structure analysis and applied it to the circular form of the IVS RNA. The native RNA was treated with dimethyl sulfate or diethyl pyrocarbonate to modify bases not involved in secondary or tertiary interactions. The RNA was then used as a template for reverse transcription. Elongation of synthetic oligodeoxynucleotide primers was found to stop (or pause) one nucleotide prior to 1-methyladenosine, 3-methylcytidine, and 7-ethoxycarbonyladenosine residues. The detection of 1-methyladenosine is particularly useful for locating single-stranded regions. After chemical cleavage of the RNA, 7-methylguanosine also could be detected. In general, the sites of modification were consistent with a previous model of the secondary structure of the linear form of the IVS RNA, a model based on enzymatic cleavage data, free energy calculations, and phylogenetic comparison. Thus, IVS RNA autocyclization does not involve major rearrangements of the secondary structure, although there is evidence for a conformational change in one region of the molecule. The methods described here should be of general use for obtaining information about structure far from the ends of RNA molecules.
The first intron of the human growth hormone gene contains a binding site for glucocorticoid receptor.
Authors: Moore D D , Marks A R , Buckley D I , Kapler G , Payvar F , Goodman H M
Published: May 2006

Glucocorticoid receptor (GCR) protein stimulates transcription from a variety of cellular genes. We show here that GCR partially purified from rat liver binds specifically to a site within the first intron of the human growth hormone (hGH) gene, approximately 100 base pairs downstream from the start of hGH transcription. GCR binding is selectively inhibited by methylation of two short, symmetrically arranged clusters of guanine residues within this site. A cloned synthetic 24-base-pair deoxyoligonucleotide containing the predicted GCR binding sequence interacts specifically with GCR. The hGH binding site shares sequence homology with a GCR binding site upstream from the human metallothionein II gene and a subset of GCR binding sites from mouse mammary tumor virus. All of these binding sites for this eukaryotic transcriptional regulatory protein show remarkable similarity in overall geometry to the binding sites for several prokaryotic transcriptional regulatory proteins.
Identification of an additional class of C3-binding membrane proteins of human peripheral blood leukocytes and cell lines.
Authors: Cole J L , Housley G A , Dykman T R , MacDermott R P , Atkinson J P
Published: May 2006

Proteins binding the third component of complement (C3) were isolated by affinity chromatography from surface-labeled solubilized membranes of human peripheral blood cells and cell lines. The isolated molecules were subjected to NaDodSO4/PAGE, and autoradiographs of these gels indicated that C3-binding proteins could be divided into three groups based on Mr: (i) gp200, an approximately 200,000 Mr molecule previously identified as the C3b/C4b receptor or CR1; (ii) gp140, an approximately 140,000 Mr molecule previously identified as the C3d receptor or CR2; and (iii) gp45-70, a heretofore unrecognized group of 45,000-70,000 Mr C3-binding molecules. The cell distribution, Mr, antigenic cross-reactivity, and specificity of gp45-70 were examined. Erythrocytes have no detectable gp45-70, but all leukocyte populations examined possess this group of molecules. On neutrophils and mononuclear phagocytes, CR1 is the predominant C3-binding glycoprotein, but gp45-70 is present on both cell populations and on macrophage and neutrophil cell lines. B plus null cells, chronic lymphocytic leukemia cells, and an Epstein-Barr virus-transformed B-cell line possess CR1, CR2, and gp45-70. On T cells and T-cell lines gp45-70 is the predominant or, in some cases, the only C3-binding protein isolated. gp45-70 is structurally characterized as a broad band or doublet with a mean Mr that is slightly different for each cell population. gp45-70 binds iC3, C3b, and C4b, but not C3d, indicating that the binding region is probably within the C3c portion of C3b. A polyclonal antibody to CR1 and monoclonal antibodies to CR1 and CR2 do not immunoprecipitate gp45-70. While gp45-70 has not been previously characterized on human cells, a C3b-binding glycoprotein of similar Mr is present on rabbit alveolar macrophages. We conclude that gp45-70 is an additional group of membrane proteins present on human leukocytes that possess ligand-binding activity for C3b.
Influence of solvent accessibility and intermolecular contacts on atomic mobilities in hemerythrins.
Authors: Sheriff S , Hendrickson W A , Stenkamp R E , Sieker L C , Jensen L H
Published: May 2006

Thermal factor parameters (B values) have been compared from the refined crystal structures of the myohemerythrin from Themiste zostericola and of the octameric hemerythrin from Themiste dyscrita. These B values, which are directly related to atomic mobilities, were found to correlate rather closely with the solvent accessible areas within the respective crystals. Although protomeric units of the two molecules have exceptionally similar three-dimensional structures, there are marked differences between the patterns of relative atomic mobilities along the polypeptide chains. The differences correspond to lattice and oligomer contacts. An adjustment of the B values based on the fraction of accessible area occluded by contacts yields values that correlate well between the independent subunits and that should pertain more closely to those for the protomer free in solution.
A widely distributed nuclear protein immunologically related to the microtubule-associated protein MAP1 is associated with the mitotic spindle.
Authors: Bonifacino J S , Klausner R D , Sandoval I V
Published: May 2006

A 280-kDa protein (p280) confined to the nucleus of interphase cells becomes associated with the mitotic spindle during cell division. p280 is immunologically related to the microtubule-associated protein MAP1, as shown by cross-reactivity with monoclonal (8D12) and polyclonal antibodies raised against MAP1. However, p280 is distinct from MAP1 as judged by its lower molecular size, proteolytic degradation products, presence in preparations of purified nuclei from which MAP1 is absent, and absence from the cytosol fraction that contains MAP1. Immunofluorescence microscopy of cells in interphase using 8D12 reveals punctate staining of the nucleus, cytoplasmic microtubules, and the microtubule organizing center. Dividing cells display strong staining of the spindle, centrioles, and mid-body. The only exception to this staining pattern is marsupial Pt k2 cells that contain p280 in the nucleus and lack MAP1. These cells exhibit fluorescent staining of the nucleus and the microtubule organizing center when in interphase, of spindle and centrioles in mitosis, and show no staining of cytoplasmic and mid-body microtubules.
Mutagenic DNA repair in Streptomyces.
Authors: Stonesifer J , Baltz R H
Published: May 2006

Streptomyces fradiae JS6 (mcr-6) is defective in the repair of potentially lethal damage to DNA induced by mitomycin C (MC), hydroxylamine (NH2OH), methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and ultraviolet light (UV), but it exhibits nearly normal sensitivity to ethyl methanesulfonate (EMS)-induced lethality. JS6 is substantially less mutable by MNNG, MMS, NQO, UV, NH2OH, and also EMS than is the parental strain. A spontaneous revertant of JS6 showed wild-type levels of resistance to all of these agents and wild-type levels of induced mutagenesis, indicating that a single mutation caused the multiple traits displayed by JS6. The mcr-6 gene product thus appears to control an error-prone (mutagenic) DNA repair system. Mediation of EMS mutagenesis by an error-prone repair pathway in S. fradiae, rather than by direct mispairing as in Escherichia coli, suggests that the streptomycetes have evolved more efficient error-avoidance mechanisms than those commonly observed in the single-celled eubacteria.
NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsules of Escherichia coli K1 and group B meningococci.
Authors: Frosch M , Görgen I , Boulnois G J , Timmis K N , Bitter-Suermann D
Published: May 2006

A system for the production of monoclonal antibodies, particularly of the IgG type, against weakly immunogenic bacterial polysaccharide antigens is described. This system, which is based on the autoimmune NZB mouse strain, has been used to produce a monoclonal IgG2a antibody against the meningococcus group B and Escherichia coli K1 polysaccharides, identical homopolymers of alpha (2----8)-linked units of N-acetylneuraminic acid that are extremely poor immunogens. Comparison of the humoral immune responses of normal BALB/c mice and autoimmune NZB mice to hyperimmunization with group A, B, and C meningococci showed that, although both strains mounted a weak meningococcal B polysaccharide-specific IgM response, only the NZB strain mounted an IgG response. Similarly, NZB mice mounted a stronger IgG response to the more immunogenic group C meningococcal polysaccharide than did BALB/c mice, although this difference was less pronounced than that observed with meningococcal B polysaccharide. No difference between the two strains of mice was demonstrable with the strongly antigenic group A meningococcal polysaccharide. These results indicate that the NZB system may be generally useful for the production of monoclonal antibodies against weakly antigenic bacterial determinants.
Depletion of mucosal mast cell protease by corticosteroids: effect on intestinal anaphylaxis in the rat.
Authors: King S J , Miller H R , Newlands G F , Woodbury R G
Published: May 2006

Rats primed by infection with the intestinal nematode Nippostrongylus brasiliensis and challenged intravenously with soluble whole-worm antigen undergo systemic anaphylactic shock. The primary lesions are in the gut and include increased permeability of the mucosa together with release, into enteric secretions, of a mucosal mast cell (MMC)-specific serine proteinase, rat mast cell protease II (RMCP-II). This enzyme is also released into the blood of shocked rats. These manifestations of anaphylaxis were abolished in rats previously treated with corticosteroids (methylprednisolone acetate, 25 mg per kg of body weight, 48 and 24 hr before i.v. challenge with antigen). Suppression of the response was associated with depletion of RMCP-II and of MMC from the intestinal mucosa. Depletion occurred 4-24 hr after treatment with as little as 1 mg of methylprednisolone per kg. By contrast, neither connective tissue mast cells nor serum levels of parasite-specific IgE were depleted in rats given 2 X 25 mg of methylprednisolone per kg. The capacity of unprimed treated rats to mount passive cutaneous anaphylaxis was, however, impaired.
Anomalous interaction of the acetylcholine receptor protein with the nonionic detergent Triton X-114.
Authors: Maher P A , Singer S J
Published: May 2006

Integral membrane proteins that form water-filled channels through membranes often exist as aggregates of similar or identical subunits spanning the membrane. It has been suggested that the insertion into the membrane of the channel-forming domains of the subunits may impart unusual structural features to the membrane-intercalated portions of the protein. To test this proposal, we have investigated the interaction of a multisubunit channel-forming integral membrane protein, the acetylcholine receptor protein, with the nonionic detergent Triton X-114. Whereas non-channel-forming integral membrane proteins that have heretofore been studied form mixed micelles with the detergent, the acetylcholine receptor was excluded from the Triton X-114 micelles. The structural implications of this result are discussed.
Note on four-particle wave functions
Authors: Pekeris C. L. , Frankowski K.
Published: May 2006

Dougall's expansion is used in the expression for the volume element in tetrahedral coordinates, thereby lifting the spherical triangle restriction on the angles at the tetrahedral vertex. Application is made to the evaluation of matrix elements for the ground state of three-electron atoms.
Stimulation of glycolysis and amino acid uptake in NRK-49F cells by transforming growth factor beta and epidermal growth factor.
Authors: Boerner P , Resnick R J , Racker E
Published: May 2006

Glycolysis in normal resting rat kidney cells (NRK-49F) was stimulated by a 2-hr exposure to transforming growth factors prior to assay. Transforming growth factor beta (TGF-beta) was effective when added alone, and further addition of epidermal growth factor (EGF) had little effect. The stimulation by TGF-beta was abolished when cycloheximide was present during the incubation, suggesting that protein synthesis is required for the effect. Incubation of the cells with 25 mM methionine abolished the stimulation of glycolysis by TGF-beta. The uptake of methylaminoisobutyrate via system A was stimulated by either TGF-beta or EGF. The greater than 3-fold stimulation of uptake by 1 ng of pure TGF-beta per ml was usually somewhat enhanced on addition of 0.5 ng of EGF per ml. Moreover, an antiserum against EGF receptor partially depressed the response to TGF-beta, suggesting some overlapping interactions of EGF and TGF-beta.
A minimal ribosomal RNA: sequence and secondary structure of the 9S kinetoplast ribosomal RNA from Leishmania tarentolae.
Authors: de la Cruz V F , Lake J A , Simpson A M , Simpson L
Published: May 2006

The portion of the Leishmania tarentolae kinetoplast maxicircle DNA encoding the 9S RNA gene was sequenced, and the 5' and 3' ends of the transcript were determined. A secondary structure for the 9S RNA was determined based on the Escherichia coli 16S model. The 610-nucleotide 9S RNA exhibits a minimal secondary structure in which all four domains of the E. coli 16S structure are preserved. Within domains, however, some stems and loops have been greatly reduced or eliminated entirely. It is presumed that these reduced domains represent the minimal essential small ribosomal RNA secondary structures necessary for a functional ribosome. Alignment of the L. tarentolae 9S rRNA sequence with the published Trypanosoma brucei 9S rRNA sequence shows a nucleotide similarity of 84% and a transversion/transition ratio of 1.66.
Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries.
Authors: Wood W I , Gitschier J , Lasky L A , Lawn R M
Published: May 2006

An oligonucleotide hybridization procedure has been developed that eliminates the preferential melting of A X T versus G X C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. This technique, which uses tetramethylammonium chloride, is especially helpful whenever a highly complex library is screened with a pool of oligonucleotide probes, which usually vary widely in base composition. The procedure can also be applied advantageously whenever an exact match to an oligonucleotide probe is desired, such as in screening for clones having as little as a single-base alteration generated by in vitro mutagenesis.
1H-Observe/13C-decouple spectroscopic measurements of lactate and glutamate in the rat brain in vivo.
Authors: Rothman D L , Behar K L , Hetherington H P , den Hollander J A , Bendall M R , Petroff O A , Shulman R G
Published: May 2006

We have used (13C)-1H NMR spectroscopy at 360.13 MHz to resolve the 13C coupled proton resonance of glutamate and lactate in the rat brain in vivo. The time required for the 13C fractional enrichment of the 4-CH2 position of brain glutamate to reach isotopic steady state was determined during a continuous infusion of D-[1-13C]glucose. Under conditions of ischemia, measurements made of the 3-CH3 of lactate in (13C)-1H NMR spectra revealed the relative contribution of brain glucose and glycogen to lactate formation. (13C)-1H NMR was 11 times more sensitive than 13C NMR for the detection of 13C in the 3-CH3 position of lactate and 6 times more sensitive for the detection of 13C in the 4-CH2 of glutamate under similar in vivo conditions.
Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes.
Authors: Hoch D H , Romero-Mira M , Ehrlich B E , Finkelstein A , DasGupta B R , Simpson L L
Published: May 2006

The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as "tunnel proteins" for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.
Adhesion to human cells by Escherichia coli lacking the major subunit of a digalactoside-specific pilus-adhesin.
Authors: Uhlin B E , Norgren M , Båga M , Normark S
Published: May 2006

Pathogenic bacteria frequently possess pili with specific binding properties that allow them to attach to epithelial tissue. In Escherichia coli, the pili associated with pyelonephritis (Pap pili) bind to digalactoside-containing glycolipids on the uroepithelium. Transposon-insertion mutants and deletion mutants of the cloned genetic determinant encoding synthesis of such digalactoside-binding Pap pili have been studied in E. coli K-12. Mutants that completely lack synthesis of the major Pap pili subunit protein, the papA gene product, and thereby no longer produce pili were shown to retain the binding specificity of intact Pap pili. Reduced expression of some of the remaining pap genes, presumably due to polarity effects from papA::Tn5 insertions, was circumvented by the use of a copy-number mutant plasmid vector. Derivatives carrying the papA-D genes produced Pap pili but did not bind to human cells. The products of the genes papE-G are essential for digalactoside-specific hemagglutination and for attachment to urinary bladder cells. The papC and papD genes presumably aid in surface localization and/or polymerization of the pili-adhesin subunits and are required for expression of pili as well as of the binding properties. Serological evidence is presented that suggests that a minor pilus component(s), presumably produced by the papE, -F, or -G gene, is the actual binding moiety in the digalactoside-specific interaction of Pap pilus-adhesin.
Assignment of the gene for Wilson disease to chromosome 13: linkage to the esterase D locus.
Authors: Frydman M , Bonné-Tamir B , Farrer L A , Conneally P M , Magazanik A , Ashbel S , Goldwitch Z
Published: May 2006

Wilson disease (WD) is an autosomal recessively inherited disorder of copper metabolism for which the basic defect is still unknown. Twenty-seven autosomal markers were investigated for linkage in a large inbred kindred with affected individuals in two generations. Also, serum copper and ceruloplasmin were measured on all available members. Close linkage (theta = 0.06) with a logarithm of odds (lod) score of 3.21 was found between the gene for WD and the esterase D locus. Efficient detection of linkage was made possible by the use of a multisibship inbred pedigree. The discovery of a polymorphic marker genetically linked to the WD locus has profound implications both for investigation of the primary gene defect and for clinical services.
Periodicity of leucine and tandem repetition of a 24-amino acid segment in the primary structure of leucine-rich alpha 2-glycoprotein of human serum.
Authors: Takahashi N , Takahashi Y , Putnam F W
Published: May 2006

The complete primary structure of the 3.1S leucine-rich alpha 2-glycoprotein (LRG) present in human plasma has been determined. This protein (Mr approximately 45,000) consists of a single polypeptide chain with one galactosamine and four glucosamine oligosaccharides attached. The polypeptide has two intrachain disulfide bonds and contains 312 amino acid residues of which 66 are leucine. The amino acid sequence can be exactly divided into 13 segments of 24 residues each, eight of which exhibit a periodic pattern in the occurrence of leucine, proline, and asparagine. The consensus sequence for the repeating tetracosapeptide unit is Pro-Xaa-Xaa-Leu-Leu-Xaa-Xaa-Xaa-X aa-Xaa-Leu-Xaa-Xaa-Leu-Xaa-Leu-Xaa-Xaa-Asn-Xaa-Leu-Xaa-Xaa-Leu. This periodicity suggests that the unique structure of LRG arose from a series of unequal crossovers of a precursor oligonucleotide sequence that encoded a building block rich in leucine. Overall, the amino acid sequence of LRG is not significantly homologous to the continuous sequence of any protein in the current data base. However, the consensus tetracosapeptide sequence shows strong homology to segments of many mitochondrial proteins, viral envelope proteins, and oncogene proteins that have a high leucine content and transmembrane domains. Tandem repetition of similar segments also occurs in apolipoproteins that have amphipathic helical potential. Prediction of the secondary structure by the Chou-Fasman rules and calculation of the hydrophilic/hydrophobic profile by several methods confirm the tandem repetition of largely hydrophobic structural units; these begin with a beta-turn that leads into an organized structure with alpha-helical or beta-sheet potential. These structural characteristics and the homology to mitochondrial proteins and apolipoproteins suggest that LRG is a membrane-derived or membrane-associated protein containing a series of domains capable of bipolar surface orientation.
Isolation of a somatic-cell mutant defective in phosphatidylserine biosynthesis.
Authors: Kuge O , Nishijima M , Akamatsu Y
Published: May 2006

Mutant clones of Chinese hamster ovary (CHO) cells defective in the base-exchange reaction of phospholipids with choline were isolated by using an in situ enzymatic assay for the reaction in cell colonies immobilized on polyester cloth. The specific activities of the choline-exchange reaction in extracts of one of the mutants (designated 64) grown at 33 degrees C and 40 degrees C were 13% and 6% of those in parental (CHO-K1) cells, respectively. The choline-exchange activity in the mutant was more thermolabile in cell extracts than that in the parent, suggesting that a mutation in the structural gene for the choline-exchange enzyme might have been induced in this mutant. In culture medium supplemented with lipoprotein-deficient serum, mutant 64 grew almost normally at 33 degrees C but divided only twice at 40 degrees C and then stopped growing. Labeling of intact cells with [32P]Pi showed that mutant 64 was also strikingly defective in the biosynthesis of phosphatidylserine at 40 degrees C but was normal at 33 degrees C. Most temperature-resistant revertants of mutant 64 exhibited nearly normal ability to synthesize phosphatidylserine at 40 degrees C and also showed choline-exchange activity similar to that in parental cells. The addition of phosphatidylserine to medium supplemented with newborn calf serum, in which mutant 64 grew more slowly than parental cells at 40 degrees C, restored the growth rate of the mutant to the parental level. Our findings suggest that the choline-exchange enzyme functions as the major route for the formation of phosphatidylserine and that the temperature-sensitive growth of mutant 64 is due to a defect in phosphatidylserine biosynthesis at 40 degrees C.
cDNA-derived amino acid sequence of the gamma subunit of GTPase from bovine rod outer segments.
Authors: Yatsunami K , Pandya B V , Oprian D D , Khorana H G
Published: May 2006

The sequence of the 74 amino acids in the gamma subunit of GTPase from bovine rod outer segments has been deduced from the cDNA sequence. Enriched GTPase mRNA was used to prepare a cDNA library in the expression vector lambda gt11. Clones encoding the gamma subunit were identified by screening the library with anti-GTPase antibodies and by cell-free translation of hybrid-selected mRNA. The longest cDNA (448 base pairs) contained the amino acid coding region for the entire gamma subunit as well as 5' and 3' noncoding regions of 67 and 159 base pairs, respectively. The 3' noncoding region contained the sequence, A-A-U-A-A-A, the presumed recognition sequence for polyadenylylation, and a 14-unit long poly(A) tract. The amino acid sequence derived for the gamma-subunit is mostly in agreement with that previously determined by McConnell and co-workers [McConnell, D. G., Kohnken, R. E. & Smith, A. J. (1984) Fed. Proc. Fed. Am. Soc. Exp. Biol. 43, 1585] and the partial sequence deduced by Van Dop and co-workers from cDNA [Van Dop, C., Medynski, D., Sullivan, K., Wu, A. M., Fung, B. K.-K. & Bourne, H. R. (1984) Biochem. Biophys. Res. Commun. 124, 250-255].
Sequences of the active-site peptides of three of the high-Mr penicillin-binding proteins of Escherichia coli K-12.
Authors: Keck W , Glauner B , Schwarz U , Broome-Smith J K , Spratt B G
Published: May 2006

The amino acid compositions of the radioactive peptides obtained from trypsin digestion of [14C]benzylpenicillin-labeled penicillin-binding proteins (PBPs) 1A, 1B, and 3 of Escherichia coli have been obtained. Complete digestion of these peptides with a combination of aminopeptidase M and carboxypeptidase Y showed that benzylpenicillin was bound to a serine residue in each of these proteins. Comparison of the compositions of the penicillin-labeled peptides with the complete amino acid sequences of PBPs 1A, 1B, and 3 showed that the acylated serine occurs near the middle of each of the proteins, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The sequence around the acylated serine of these high Mr PBPs shows little similarity to that around the acylated serine of the low-Mr PBPs (D-alanine carboxypeptidases) or of the class A or class C beta-lactamases, except that in all of these enzymes which interact with penicillin the acylated serine residue occurs within the sequence Ser-Xaa-Xaa-Lys.
1H NMR studies of electron exchange rate of Pseudomonas aeruginosa azurin.
Authors: Uğurbil K , Mitra S
Published: May 2006

T1 values of the His-35 C-2 proton resonance of reduced Pseudomonas aeruginosa azurin were determined at 25 degrees C and pH values 4.5, 7.3, and 9.0 in the presence of different fractional amounts of oxidized azurin. The C-2 proton of His-35 undergoes very rapid spin relaxation in oxidized azurin because of its close proximity to the paramagnetic copper. In the presence of oxidized protein, the T1 values of this proton in reduced azurin depend on the lifetime of the reduced protein. From the T1 data, the electron self-exchange rate constant for azurin was calculated to be 1.4 X 10(4) M-1 X s-1, 4.3 X 10(3) M-1 X s-1, and 6.0 X 10(3) M-1 X s-1 at pH values 4.5, 7.3, and 9, respectively. At pH 7.3, the C-2 proton of His-35 is in slow exchange between the imidazole and imidazolium forms and gives rise to two separate resonances at 9.39 and 8.00 ppm. By using these two resonances, the electron self-exchange rate constants were determined separately for the two species of azurin for which the His-35 residue is in the imidazole or the imidazolium forms; results showed that both species participate in self-exchange of electrons with equal efficiency.
The all-or-none role of innervation in expression of apamin receptor and of apamin-sensitive Ca2+-activated K+ channel in mammalian skeletal muscle.
Authors: Schmid-Antomarchi H , Renaud J F , Romey G , Hugues M , Schmid A , Lazdunski M
Published: May 2006

The long-lasting after-hyperpolarization(s) (AHP) that follows the action potential in rat myotubes differentiated in culture is due to Ca2+-activated K+ channels. These channels have the property to be specifically blocked by the bee venom toxin apamin at low concentrations. Apamin has been used in this work to analyze, by electrophysiological and biochemical techniques, the role of innervation in expression of these important channels. The main results are as follows: (i) Long-lasting AHP that follows the action potential in rat myotubes in culture disappears when myotubes are cocultured with nerve cells from the spinal cord under the conditions of in vitro innervation. (ii) Extensor digitorum longus muscles from adult rats have action potentials that are not followed by AHP but AHP are systematically recorded after muscle denervation and they are blocked by apamin. (iii) Specific 125I-labeled apamin binding is undetectable in innervated muscle fibers but it becomes detectable 2-4 days after muscle denervation to be maximal 10 days after denervation. (iv) Apamin receptors detected with 125I-labeled apamin are present at fetal stages with biochemical characteristics identical to those found in myotubes in culture. The receptor number decreases as maturation proceeds and 125I-labeled apamin receptors completely disappear after the first week of postnatal life, in parallel with the disappearance of multi-innervation. All these results taken together strongly suggest an all-or-none effect of innervation on the expression of apamin-sensitive Ca2+-activated K+ channels.
Adenovirus EIIA early promoter: transcriptional control elements and induction by the viral pre-early EIA gene, which appears to be sequence independent.
Authors: Murthy S C , Bhat G P , Thimmappaya B
Published: May 2006

A molecular dissection of the adenovirus EIIA early (E) promoter was undertaken to study the sequence elements required for transcription and to examine the nucleotide sequences, if any, specific for its trans-activation by the viral pre-early EIA gene product. A chimeric gene in which the EIIA-E promoter region fused to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene was used in transient assays to identify the transcriptional control regions. Deletion mapping studies revealed that the upstream DNA sequences up to -86 were sufficient for the optimal basal level transcription in HeLa cells and also for the EIA-induced transcription. A series of linker-scanning (LS) mutants were constructed to precisely identify the nucleotide sequences that control transcription. Analysis of these LS mutants allowed us to identify two regions of the promoter that are critical for the EIIA-E transcription. These regions are located between -29 and -21 (region I) and between -82 and -66 (region II). Mutations in region I affected initiation and appeared functionally similar to the "TATA" sequence of the commonly studied promoters. To examine whether or not the EIIA-E promoter contained DNA sequences specific for the trans-activation by the EIA, the LS mutants were analyzed in a cotransfection assay containing a plasmid carrying the EIA gene. CAT activity of all of the LS mutants was induced by the EIA gene in this assay, suggesting that the induction of transcription of the EIIA-E promoter by the EIA gene is not sequence-specific.
Purification and characterization of two cartilage-inducing factors from bovine demineralized bone.
Authors: Seyedin S M , Thomas T C , Thompson A Y , Rosen D M , Piez K A
Published: May 2006

Two naturally occurring peptides that induce chondrogenesis in culture have been purified to apparent homogeneity. These cartilage-inducing factors (CIF-A and CIF-B) were isolated from bovine demineralized bone by dissociative extraction, gel filtration, cation-exchange chromatography, and reversed-phase HPLC. CIF-A and CIF-B at concentrations of 1-10 ng/ml each induce embryonic rat mesenchymal cells in culture to assume a cartilage morphology and synthesize cartilage-specific proteoglycan and type II collagen. The amino acid compositions of CIF-A and CIF-B are similar but not identical. Both factors have an apparent Mr of 26,000, as determined by NaDodSO4/PAGE. In the presence of 2-mercaptoethanol, both are converted to species of about one-half that Mr, indicating that they are dimers of identical or very similar chains.
A method for mapping intranuclear protein-DNA interactions and its application to a nuclease hypersensitive site.
Authors: Jackson P D , Felsenfeld G
Published: May 2006

We have devised a method for mapping sites on DNA within the nucleus that are protected against nuclease attack by interaction with bound protein or other factors. This "footprinting" method uses an end-labeled sequence-specific DNA probe, which is annealed to the DNA from nuclear digests under carefully controlled conditions. The annealed complexes are treated with single-strand-specific nuclease, the resulting duplex molecules are electrophoresed on gels, and the gels are autoradiographed. The high sensitivity and resolution of the method have made it possible to obtain a detailed map of DNase I cutting patterns in the 5' flanking sequence of the chicken adult beta (beta A)-globin gene within nuclei from various tissues. In nuclei from adult erythrocytes, this domain is hypersensitive to nucleases. However, we detect within the domain two well-defined regions that are protected against attack, indicating the presence of one or more bound factors. Nuclei from oviduct or 5-day-old embryonic erythrocytes, in which the domain is not hypersensitive, show limited and different patterns of protection.
Identification of cDNA clones encoding a precursor of rat liver cathepsin B.
Authors: San Segundo B , Chan S J , Steiner D F
Published: May 2006

Recent studies have suggested that many lysosomal enzymes, including cathepsin B (EC 3.4.22.1), may be synthesized as larger precursors and proteolytically processed to their mature forms. To determine the structure of the primary translation product of cathepsin B, we have screened a phage cDNA library for clones encoding rat liver cathepsin B. We synthesized two extended DNA oligonucleotides to use as hybridization probes: a 50-mer corresponding to the coding segment for residues 215-231 of mature cathepsin B and a 54-mer corresponding to residues 117-134. After screening 600,000 plaques, five clones were obtained that hybridized to the 32P-labeled 50-mer; of these, two (lambda rCB3 and lambda rCB5) also reacted with the 54-mer. DNA sequence analysis confirmed that lambda rCB3 and lambda rCB5 both encoded rat liver cathepsin B, and the translated sequence is in agreement with the sequence determined [Takio, K., Towatari, T., Katunuma, N., Teller, D. C. & Titani, K. (1983) Proc. Natl. Acad. Sci. USA 80, 3666-3670], except for a tryptophan for glycine substitution at residue 78 and the presence of two amino acids at the junction site of the light and heavy chains. Moreover, the DNA sequence reveals an open reading frame extending beyond the 5' (NH2 terminus), and the predicted COOH terminus of the coding sequence for the mature protein is extended by six amino acids. These results confirm that the biosynthesis of cathepsin B involves a larger precursor form and demonstrate the effectiveness of long oligonucleotide probes for screening to detect rare cloned mRNAs.
Lens-specific expression of the chloramphenicol acetyltransferase gene promoted by 5' flanking sequences of the murine alpha A-crystallin gene in explanted chicken lens epithelia.
Authors: Chepelinsky A B , King C R , Zelenka P S , Piatigorsky J
Published: May 2006

We have developed a system using explanted embryonic chicken lens epithelia to express foreign recombinant genes containing crystallin DNA regulatory sequences introduced by calcium phosphate transfection. Optimal results were obtained with lens epithelia from 14-day embryos transfected 1 day after explantation and assayed 3 days later. When DNA sequences (-364 to +45) of the murine alpha A-crystallin gene were inserted in the pSVO-CAT expression vector of Gorman et al. [Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051] in the same orientation as in the crystallin gene, they promoted chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) activity in the transfected epithelia. Sequences 87 to 364 base pairs upstream from the murine gene cap site were required for CAT gene expression. These crystallin gene regulatory sequences did not promote CAT expression in primary cultures of embryonic chicken fibroblasts or other nonlens cells. By contrast, the long terminal repeat of Rous sarcoma virus and the early promoter of simian virus 40 promoted CAT activity in lens and nonlens cells. Our experiments thus demonstrate that the explanted embryonic chicken lens epithelium is an advantageous recipient for identifying lens-cell-specific regulatory sequences of crystallin genes and implicate a DNA region upstream of the "TATA box" for regulation of the murine alpha A-crystallin gene. These experiments also suggest that explanted epithelia from other tissues may be useful for studying the expression of foreign genes.
Evolution and organization of the fibrinogen locus on chromosome 4: gene duplication accompanied by transposition and inversion.
Authors: Kant J A , Fornace A J , Saxe D , Simon M I , McBride O W , Crabtree G R
Published: May 2006

Human fibrinogen cDNA probes for the alpha-, beta-, and gamma-polypeptide chains have been used to isolate the corresponding genes from human genomic libraries. There is a single copy of each gene. Restriction endonuclease analysis of isolated genomic clones and human genomic DNA indicates that the human alpha-, beta-, and gamma-fibrinogen genes are closely linked in a 50-kilobase region of a single human chromosome: the alpha-gene in the middle flanked by the beta-gene on one side and the gamma-gene on the other. The alpha- and gamma-chain genes are oriented in tandem and transcribed toward the beta-chain gene. The beta-chain gene is transcribed from the opposite DNA strand toward the gamma- and alpha-chain genes. The three genes have been localized to the distal third of the long arm of chromosome 4, bands q23-q32, by in situ hybridization with fibrinogen cDNAs and by examination of DNA from multiple rodent-human somatic cell hybrids. Alternative explanations for the present arrangement of the three fibrinogen genes involve either a three-step mechanism with inversion of the alpha/gamma-region or a two-step mechanism involving remote transposition and inversion. The second more simple mechanism has a precedent in the origin of repeated regions of the fibrinogen and immunoglobulin genes.
Nature of the charged-group effect on the stability of the C-peptide helix.
Authors: Shoemaker K R , Kim P S , Brems D N , Marqusee S , York E J , Chaiken I M , Stewart J M , Baldwin R L
Published: May 2006

The residues responsible for the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A) have been identified by chemical synthesis of analogues and measurement of their helix-forming properties. Each of the residues ionizing between pH 2 and pH 8 has been replaced separately by an uncharged residue. Protonation of Glu-2- is responsible for the sharp decrease in helix stability between pH 5 and pH 2, and deprotonation of His-12+ causes a similar decrease between pH 5 and pH 8. Glu-9- is not needed for helix stability. The results cannot be explained by the Zimm-Bragg model and host-guest data for alpha-helix formation, which predict that the stability of the C-peptide helix should increase when Glu-2- is protonated or when His-12+ is deprotonated. Moreover, histidine+ is a strong helix-breaker in host-guest studies. In proteins, acidic and basic residues tend to occur at opposite ends of alpha-helices: acidic residues occur preferentially near the NH2-terminal end and basic residues near the COOH-terminal end. A possible explanation, based on a helix dipole model, has been given [Blagdon, D. E. & Goodman, M. (1975) Biopolymers 14, 241-245]. Our results are consistent with the helix dipole model and they support the suggestion that the distribution of charged residues in protein helices reflects the helix-stabilizing propensity of those residues. Because Glu-9 is not needed for helix stability, a possible Glu-9-...His-12+ salt bridge does not contribute significantly to helix stability. The role of a possible Glu-2-...Arg-10+ salt bridge has not yet been evaluated. A charged-group effect on alpha-helix stability in water has also been observed in a different peptide system [Ihara, S., Ooi, T. & Takahashi, S. (1982) Biopolymers 21, 131-145]: block copolymers containing (Ala)20 and (Glu)20 show partial helix formation at low temperatures, pH 7.5, where the glutamic acid residues are ionized. (Glu)20(Ala)20Phe forms a helix that is markedly more stable than (Ala)20(Glu)20Phe. The results are consistent with a helix dipole model.
Renaturation of skeletal muscle tropomyosin: implications for in vivo assembly.
Authors: Brown H R , Schachat F H
Published: May 2006

The observation that the alpha beta heterodimer is the predominant species of tropomyosin in rabbit skeletal muscles has led to the suggestion that this species assembles preferentially. To understand the molecular basis of this assembly process, we have studied renaturation under conditions that favor heterodimer formation. When skeletal muscle tropomyosin composed of equal amounts of alpha and beta subunits is renatured either by cooling or by dialysis a distribution that favors homodimers is generated. In contrast, rapid renaturation by dilution from urea favors the heterodimer. Further analysis of this latter renaturation procedure with cysteine-cleavage fragments of tropomyosin using circular dichroic measurements shows that as few as 30 residues in the NH2-terminal third of each tropomyosin subunit are involved in the initial interaction that results in heterodimer formation. Based on the density of sequence substitutions between the alpha and beta subunits, that region probably includes residues 36-64.
Interferon stimulates cholesterol and phosphatidylcholine synthesis but inhibits cholesterol ester synthesis in HeLa-S3 cells.
Authors: Pfeffer L M , Kwok B C , Landsberger F R , Tamm I
Published: May 2006

Treatment of human HeLa-S3 cells (an epidermoid carcinoma line) with human beta-interferon (640 units/ml) selectively alters lipid metabolism by increasing cholesterol synthesis per mg of cell protein as measured by 1-hr pulse-labeling of cells with [3H]acetate. Cholesterol synthesis in interferon-treated cells is increased approximately equal to 60% at 24 hr after the beginning of treatment and approximately equal to 450% at 48 hr. Continuous labeling of interferon-treated cells with [14C]acetate shows increased accumulation of label in cholesterol when normalized per mg of cell protein, as well as an increase in the specific activity of cholesterol in the treated cells. In contrast, interferon treatment decreases the accumulation of [14C]acetate into cholesterol esters. The [14C]acetate labeling of sphingomyelin, phosphatidylethanolamine, and triglycerides shows no change compared to untreated controls. The labeling of phosphatidylcholine was moderately increased in treated cells. The interferon-induced changes in lipid metabolism are a part of a coordinated response of cells to interferon treatment, characterized by reduced cell proliferation and cell motility and an increase in cell size and mass. The increased cholesterol synthesis is consistent with a model in which beta-interferon treatment of HeLa cells inhibits the endocytosis of cholesterol-containing low density lipoprotein, which results in an increase in cholesterol synthesis.