bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantum simulation of nuclear rearrangement in electron transfer reactions
Authors: Zheng Chong , McCammon J. Andrew , Wolynes Peter G.
Published: May 2006

A quantum simulation scheme based on the path integral molecular dynamics technique has been used to calculate the effective activation energies associated with nuclear rearrangement in the electron transfer reactions Co(NH
3
)
6
2+
+ Co(NH
3
)
6
3+
→ Co(NH
3
)
6
3+
+ Co(NH
3
)
6
2+
and Ru(NH
3
)
6
2+
+ Ru(NH
3
)
6
3+
→ Ru(NH
3
)
6
3+
+ Ru(NH
3
)
6
2+
. Even with a simple Hamiltonian and short time dynamic simulations, the results are in satisfactory agreement with other theoretical calculations. This simulation approach can be used in chemical and biological systems where the reactions are largely controlled by nuclear rearrangements, such as those of electron transfer reactions in some electron carrier proteins.
Production of simian virus 40 large tumor antigen in bacteria: altered DNA-binding specificity and dna-replication activity of underphosphorylated large tumor antigen.
Authors: Mohr I J , Gluzman Y , Fairman M P , Strauss M , McVey D , Stillman B , Gerard R D
Published: May 2006

A bacterial expression system was used to produce simian virus 40 large tumor antigen (T antigen) in the absence of the extensive posttranslational modifications that occur in mammalian cells. Wild-type T antigen produced in bacteria retained a specific subset of the biochemical activities displayed by its mammalian counterpart. Escherichia coli T antigen functioned as a helicase and bound to DNA fragments containing either site I or the wild-type origin of replication in a manner identical to mammalian T antigen. However, T antigen purified from E. coli did not efficiently bind to site II, an essential cis element within the simian virus 40 origin of replication. It therefore could not unwind origin-containing plasmids or efficiently replicate simian virus 40 DNA in vitro. The ability of protein phosphorylation to modulate the intrinsic preference of full-length T antigen for either site I or site II is discussed.
Mammalian multidrug-resistance gene: correlation of exon organization with structural domains and duplication of an ancestral gene.
Authors: Raymond M , Gros P
Published: May 2006

Analysis of the nucleotide and deduced amino acid sequences of the biologically active mouse mdr1 cDNA clone indicates that the protein is formed by two highly homologous halves, each containing six putative transmembrane domains and a nucleotide-binding site. The duplicated unit shows high sequence homology to the proposed energy-coupling subunit of bacterial periplasmic transport proteins. We have cloned and characterized the mouse mdr1 gene and have analyzed the genomic organization of the two homologous halves forming the mdr1 protein. The gene spans 68 kilobases, is split into 28 exons, and the two homologous halves are encoded by 14 and 13 exons. The transcriptional initiation site of the gene has been mapped and putative TATA and consensus CAAT sequences have been found at positions -27 and -83, respectively. Discrete structural domains of the mdr1 protein are encoded by separate exons: Ten of the 12 putative transmembrane domains are encoded by individual exons and the two nucleotide-binding sites are each encoded by three exons. The exon/intron organization of the gene is conserved in the two highly homologous regions encoding the nucleotide-binding sites. The conservation of certain pairs of introns, together with the high degree of sequence homology, indicate that the mouse mdr1 gene originated from the duplication of an intron-containing ancestral gene.
Pathway of proton transfer in bacterial reaction centers: replacement of glutamic acid 212 in the L subunit by glutamine inhibits quinone (secondary acceptor) turnover.
Authors: Paddock M L , Rongey S H , Feher G , Okamura M Y
Published: May 2006

The mechanism of proton transfer in the reaction centers (RCs) from Rhodobacter sphaeroides was investigated by site-directed mutagenesis. Replacement of Glu-212 of the L subunit, a protonatable residue located near the secondary acceptor (QB) binding site, by glutamine reduced the in vitro electron turnover from cytochrome c to 2,3-dimethoxy-5-methylbenzoquinone (UQ0) by a factor of 25. The electron transfer rate to QB remained essentially unimpaired. Consequently, it is postulated that the reduced turnover in the mutant is due to a reduced rate of proton transfer to QB2-. The lack of pH dependence of the forward electron transfer rate DQA-QB----DQAQB- and the back reaction rate D+QAQB- ----DQAQB (where D = primary donor and QA = primary acceptor) in the mutant RC indicate that the observed pH dependence in the native RC is due to Glu-212, which has an anomalously high pKa value of 9.5 +/- 0.3. These results support the involvement of Glu-212 as a proton donor to reduced QB.
Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.
Authors: Hubbard B R , Ulrich M M , Jacobs M , Vermeer C , Walsh C , Furie B , Furie B C
Published: May 2006

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamic acid residues to form gamma-carboxyglutamic acid residues within the vitamin K-dependent proteins. This enzyme recognizes the gamma-carboxylation recognition site on the propeptide of the precursor forms of the vitamin K-dependent blood coagulation proteins. To purify this enzyme to homogeneity, the carboxylase from bovine liver microsomes was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), the protein was fractionated with ammonium sulfate, and then the enzyme was isolated by affinity chromatography using a synthetic peptide based upon the structure of the prothrombin propeptide. Elution with 10 mM propeptide yielded a single major band on SDS gel electrophoresis with a molecular weight of 77,000. In the presence of high concentrations of propeptide, only minimal carboxylase activity was measurable. Antibodies to the protein inhibited the carboxylase activity in crude preparations. In an alternative affinity purification strategy the propeptide was coupled through an NH2-terminal cysteine to an activated thiol-Sepharose column. The carboxylase-propeptide complex was eluted at 25 degrees C by reductive cleavage of the enzyme-propeptide complex in the presence of detergent and phospholipids. The eluted protein (Mr, 77,000) contained both stable vitamin K-dependent carboxylase and vitamin K epoxidase activity. The protein, purified by either method, was detected as a single band (Mr, 77,000) in a Western blot using anti-carboxylase antibodies. A 10,000-fold purification of carboxylase activity from crude microsomes was estimated. Purified bovine liver vitamin K-dependent carboxylase should facilitate the study of its structure and of the mechanism of action of vitamin K as a cofactor in the reaction catalyzed by this enzyme.
Photogating of ionic currents across a lipid bilayer.
Authors: Drain C M , Christensen B , Mauzerall D
Published: May 2006

Photoformation of metalloporphyrin cations in a lipid bilayer increases the ionic currents of negative and decreases those of positive hydrophobic ions. At low concentrations of the mobile hydrophobic ion, a 30% change in conductivity is observed that decreases with increasing concentration of positive tetraphenylphosphonium ion and increases drastically with increasing concentration of negative tetraphenylboride ion. In the region of saturated conductance of boride ion, the increase in conductivity is 3.6-fold. A 15-fold increase is observed with the protonophore carbonyl cyanide 3-chlorophenylhydrazone. In this case the net charge gated is 300 times greater than the photogenerated charge in the bilayer membrane. Thus there is a net gain in this organic field effect phototransistor. The gating can also be accomplished by continuous light or chemical oxidants. Photogating is explained as space charge effects inside the bilayer.
Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA.
Authors: Behr J P , Demeneix B , Loeffler J P , Perez-Mutul J
Published: May 2006

A general and efficient transfection procedure, based on compacted lipopolyamine-coated plasmids, has been developed. The active species is obtained by simple addition of excess synthetic lipospermine solution to the DNA and binds within minutes to the cell membrane. This technique has been developed on endocrine cells of the intermediate lobe of the pituitary as a general tool for physiological work on primary cells; it is not toxic and does not interfere with physiological regulations in melanotrope cells. A variety of eukaryotic cell cultures also have been transfected with success for transient and stable expression.
Wounding induces the first enzyme of the shikimate pathway in Solanaceae
Authors: Dyer William E. , Henstrand John M. , Handa Avtar K. , Herrmann Klaus M.
Published: May 2006

The first enzyme of the shikimate pathway, 3-deoxy-D-
arabino
-heptulosonate-7-phosphate synthase (EC 4.1.2.15), is induced by wounding potato or tomato tissue. The increase in enzyme activity is associated with elevated amounts of the enzyme as determined by immunoblots. The specific wound-induced protein synthesis is preceded by an increase in the mRNA encoding this enzyme. The induced mRNA of potato tuber, leaf, and stem tissue is translated into a precursor polypeptide that is recognized by antibodies raised against the mature enzyme from tuber plastids. Wounding also induces mRNA encoding phenylalanine ammonia-lyase (EC 4.3.1.5), a key enzyme of plant secondary metabolism. The time courses for the induction of the two enzymes are similar, suggesting coordinate regulation for the biosynthesis of primary and secondary aromatic compounds.
Hemopoietic stem cells in murine embryonic yolk sac and peripheral blood.
Authors: Toles J F , Chui D H , Belbeck L W , Starr E , Barker J E
Published: May 2006

Disaggregated embryonic yolk sac cells and circulating peripheral blood cells were obtained from normal murine day 9 embryos, prior to the formation of the fetal liver. These cells were microinjected transplacentally into days 11-15 W mutant anemic fetuses, when the fetal liver was the major hemopoietic organ. In a small proportion of the recipient animals examined after birth, long-term repopulation by the embryonic donor hemopoietic cells was observed. The donor hemopoietic stem cells proliferated and differentiated in the hosts as evidenced by the presence of donor hemoglobins in the growing recipient host animals. Some mothers of the pups were also repopulated by the donor stem cells. These results provide direct evidence that, during early murine embryogenesis, there are functional hemopoietic stem cells which are capable of colonizing the adult hemopoietic organs and probably the fetal liver and spleen to initiate hemopoiesis in these tissues.
Provirus insertion in Tpl-1, an Ets-1-related oncogene, is associated with tumor progression in Moloney murine leukemia virus-induced rat thymic lymphomas.
Authors: Bear S E , Bellacosa A , Lazo P A , Jenkins N A , Copeland N G , Hanson C , Levan G , Tsichlis P N
Published: May 2006

T-cell lymphomas induced in rats by Moloney murine leukemia virus acquire increasing numbers of proviruses in their genome during tumor progression in vivo and passage of tumor cells in vitro. To determine whether the proviruses progressively acquired during tumor progression play a causal role in this process, we cloned one of them from a cell line derived from the primary tumor 2772. A probe from the cellular DNA flanking the provirus was used to analyze 79 DNA samples from primary tumor tissues of 28 tumor-bearing rats and 80 DNA samples from 30 independent tumor cell lines. This analysis revealed a rearrangement in this region in the primary tumor derived from the thymus of one animal but not in a clone of the same tumor segregating in the spleen. Of the cell line DNA samples, three carried a provirus in this region. Two of these integration events had occurred independently in two clonally related sublines derived from tumor 2772, and they were followed by rapid selection in culture. On the basis of these findings this locus was named Tpl-1 (tumor progression locus 1). The Tpl-1 locus was mapped to rat chromosome 8 and to mouse chromosome 9 at a genetic distance of 1.2 +/- 0.9 centimorgans from the Ets-1 protooncogene. Although the genetic distance between Tpl-1 and Ets-1 indicates that they are different genes, analysis of Tpl-1 cDNA clones revealed that the two are closely related.
Mutations in the structural genes for eukaryotic initiation factors 2 alpha and 2 beta of Saccharomyces cerevisiae disrupt translational control of GCN4 mRNA.
Authors: Williams N P , Hinnebusch A G , Donahue T F
Published: May 2006

The SUI2 and SUI3 genes of Saccharomyces cerevisiae encode the alpha and beta subunits, respectively, of translation initiation factor eIF-2 (eukaryotic initiation factor 2). Previously isolated mutations in these genes restore expression from his4 mutant alleles lacking an ATG initiation codon. The SUI mutations also lead to increased levels of HIS4 mRNA. We show that the latter phenotype exists because the SUI mutations elevate expression of GCN4, an activator of HIS4 transcription. Increased GCN4 expression in the SUI mutants occurs independently of the GCN2 and GCN3 gene products that are normally required to stimulate translation of GCN4 mRNA under conditions of amino acid starvation. Derepression of GCN4 expression in the SUI mutants requires the multiple AUG codons in the leader of the GCN4 transcript that normally mediate its translational control by amino acid availability. In these respects, the SUI mutations resemble mutations in GCD genes whose products function as translational repressors of GCN4. Thus, in addition to its general role in AUG start codon selection, eIF-2 appears to be an important factor in GCN4 translational control. We also show that deletion of GCN3 in sui2-1 strains is lethal, suggesting that GCN3 contributes to eIF-2 alpha function in addition to its role as a translational activator of GCN4.
Molecular characterization of a 23-kilodalton major antigen secreted by Toxoplasma gondii.
Authors: Cesbron-Delauw M F , Guy B , Torpier G , Pierce R J , Lenzen G , Cesbron J Y , Charif H , Lepage P , Darcy F , Lecocq J P
Published: May 2006

The strategy chosen for cloning potential vaccine antigens of Toxoplasma gondii was based on the hypothesis that the definitive protection observed in natural infection is due to the presence of encysted bradyzoite forms in host tissues throughout life. The antigens released by the bradyzoites would maintain an immune response against the invading tachyzoites. This led us to identify in tachyzoite in vitro translation products a polypeptide of 24 kDa that is an excreted-secreted antigen (ESA) and is cross-reactive with bradyzoites. In addition, the detection of anti-P24 IgG antibodies is correlated with the chronic infection in man. The gene encoding P24 has been isolated, sequenced, and expressed in Escherichia coli and eukaryotic cells. The recombinant proteins were immunogenic in mice, producing anti-native P23 antibodies. Immunocytochemical analysis located the native antigen in the dense granules of both tachyzoite and bradyzoite forms and showed that it is secreted within host-cell-modified phagosome. Moreover 45Ca2+ labeling as well as regional homologies indicate that this protein has Ca2+-binding properties, suggesting its physiological importance in host-cell invasion. P23 is of diagnostic interest as a marker of chronic toxoplasmosis and is proposed as a vaccine component.
Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells.
Authors: Goldgaber D , Harris H W , Hla T , Maciag T , Donnelly R J , Jacobsen J S , Vitek M P , Gajdusek D C
Published: May 2006

We have analyzed the modulation of amyloid beta-protein precursor (APP) gene expression in human umbilical vein endothelial cells (HUVEC). The level of the APP mRNA transcripts increased as HUVEC reached confluency. In confluent culture the half-life of the APP mRNA was 4 hr. Treatment of the cells with human-recombinant interleukin 1 (IL-1), phorbol 12-myristate 13-acetate, or heparin-binding growth factor 1 enhanced the expression of APP gene in these cells, but calcium ionophore A23187 and dexamethasone did not. The protein kinase C inhibitor 1-(isoquinolinsulfonyl)-2-methylpiperazine (H7) inhibited IL-1-mediated increase of the level of APP transcripts. To map IL-1-responsive elements of the APP promoter, truncated portions of the APP promoter were fused to the human growth hormone reporter gene. The recombinant plasmids were transfected into mouse neuroblastoma cells, and the cell medium was assayed for the human growth hormone. A 180-base-pair region of the APP promoter located between position -485 and -305 upstream from the transcription start site was necessary for IL-1-mediated induction of the reporter gene. This region contains the upstream transcription factor AP-1 binding site. These results suggest that IL-1 upregulates APP gene expression in HUVEC through a pathway mediated by protein kinase C, utilizing the upstream AP-1 binding site of the APP promoter.
Expression of Drosophila Shaker potassium channels in mammalian cells infected with recombinant vaccinia virus.
Authors: Leonard R J , Karschin A , Jayashree-Aiyar S , Davidson N , Tanouye M A , Thomas L , Thomas G , Lester H A
Published: May 2006

A recombinant vaccinia virus containing a Drosophila potassium channel (Shaker H4) cDNA was constructed by homologous recombination between wild-type vaccinia virus DNA and a transfer plasmid. The new virus was used to infect four types of mammalian cells in culture. Electrophysiological recording 24-72 hr after infection revealed the expression of voltage-gated transient potassium channels in all four cell types. The properties of the induced currents were identical to those previously observed following injection of the Shaker H4 transcript into oocytes. Vaccinia promises to be an effective vehicle for the heterologous expression of transmembrane ion channels in a variety of cell types.
In vivo DNA loops in araCBAD: size limits and helical repeat.
Authors: Lee D H , Schleif R F
Published: May 2006

Formation of a DNA loop by AraC proteins bound at the araI and araO2 sites, whose center-to-center distance is 211 base pairs, is necessary for repression of the araBAD promoter, PBAD, of Escherichia coli. To determine the upper and lower size limits of the loop, we constructed PBAD-reporter gene fusion plasmids with various spacings between araI and araO2 and measured their levels of expression. Spacings larger than about 500 base pairs resulted in elimination of detectable repression. No lower limit to spacing was found, suggesting that AraC protein itself possesses significant flexibility and its bending substantially aids formation of small loops. As the spacing between araI and araO2 varied, the activity of PBAD oscillated with an 11.1-base-pair periodicity, implying that the in vivo helical repeat of this DNA is 11.1 base pairs per turn.
Increased clearance of plasma cholesterol after injection of apolipoprotein E into Watanabe heritable hyperlipidemic rabbits.
Authors: Yamada N , Shimano H , Mokuno H , Ishibashi S , Gotohda T , Kawakami M , Watanabe Y , Akanuma Y , Murase T , Takaku F
Published: May 2006

Apolipoprotein E (apoE) is known to play an important role in lipoprotein metabolism. We have studied the effect of apoE on the metabolism of plasma cholesterol by injecting apoE intravenously into rabbits deficient in low density lipoprotein receptors [Watanabe heritable hyperlipidemic (WHHL) rabbits]. Approximately 30 mg of apoE was injected per rabbit; a total of five WHHL rabbits were used. One hour later, plasma cholesterol levels fell 8.3% (from 488 +/- 192 to 446 +/- 174 mg/dl). After 3 hr, cholesterol levels had fallen by 19% (to 392 +/- 152 mg/dl). The reduced levels were maintained for at least 8 hr after injection of apoE. Cholesterol in very low density lipoproteins (VLDLs) and intermediate density lipoproteins fell rapidly during the first 2 hr after injection, followed by a reduction in the low density lipoprotein cholesterol level. Changes in apolipoprotein B levels in each lipoprotein fraction were very similar to those of cholesterol. Plasma apoE levels 3 min after injection were elevated 3-fold to 22.8 +/- 6.3 mg/dl and returned to initial levels 8 hr after injection. The rate of removal of intravenously injected 125I-labeled VLDL that had been incubated with apoE was 3-fold higher than that of unmodified VLDL. From these results, we conclude that the injected apoE is incorporated into VLDLs and that VLDL particles carrying more apoE are removed from the blood more rapidly, resulting in reduced formation of low density lipoprotein and lowered cholesterol levels.
Cell-surface expression and purification of human CD4 produced in baculovirus-infected insect cells.
Authors: Webb N R , Madoulet C , Tosi P F , Broussard D R , Sneed L , Nicolau C , Summers M D
Published: May 2006

CD4 is an integral membrane glycoprotein that acts as the cellular receptor for human immunodeficiency virus (HIV). A cDNA encoding full-length CD4 was inserted into the genome of Autographa californica nuclear polyhedrosis virus under transcriptional regulation of the viral polyhedrin gene promoter. The recombinant virus was used to infect insect cells, which resulted in the abundant expression of CD4 as evaluated by flow cytometry and immunoblot analysis. Recombinant CD4 expressed on the surface of infected insect cells was immunologically indistinguishable from human CD4 when using 11 different anti-CD4 monoclonal antibodies. The extraction of infected cells by phase-transition separation with Triton X-114 followed by immunoaffinity chromatography yielded a single protein detected by NaDodSO4/PAGE using silver staining. N-terminal sequence analysis of the purified recombinant protein showed that CD4 produced in Sf9 cells is efficiently cleaved from the precursor protein. Immunoblot analysis under nondenaturing conditions showed that the purified protein reacted with the anti-CD4 monoclonal antibody Leu-3a. The potential use of the recombinant membrane-associated CD4 in anti-HIV therapy is discussed.
Stimulation of beta-adrenergic receptors of S49 lymphoma cells redistributes the alpha subunit of the stimulatory G protein between cytosol and membranes.
Authors: Ransnäs L A , Svoboda P , Jasper J R , Insel P A
Published: May 2006

The stimulatory guanine nucleotide-binding protein (Gs), which links cell-surface receptors to second-messenger effector systems, is assumed to be confined to plasma membranes. In the current studies we tested whether Gs redistributes within cells by treating S49 lymphoma cells with the beta-adrenergic agonist isoproterenol, then separating cytosol and crude membrane fractions (defined as pellet and supernatant, respectively, after centrifugation for 1 hr at 150,000 x g), and assaying fractions for the alpha subunit of Gs (alpha s) using a competitive ELISA and reconstitution techniques. Under basal conditions, a small (10%) pool of alpha s was identified in supernatant fractions of S49 cells. The size of this pool decreased in the first 15 min after agonist treatment of cells. This decrease was blocked by a beta-adrenergic receptor antagonist and did not occur in an S49 variant, UNC, which lacks functional interaction between receptors and Gs. The size of the alpha s pool in supernatant fractions increased to almost 50% of total cellular alpha s during a 1-hr incubation of cells with isoproterenol. Before isoproterenol treatment only the competitive ELISA was sensitive enough to detect cytosolic alpha s, whereas at later time points (greater than or equal to 30 min) the presence of alpha s in the cytosol was confirmed by both immunoblotting and by reconstitution of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in Gs-deficient membranes derived from cyc-S49 cells. In contrast to membrane alpha s, cytosolic alpha s did not require activation (e.g., by AlF4-) in the reconstitution assay to stimulate adenylyl cyclase. Use of an antibody that selectively recognizes monomeric dissociated alpha s, but not heterotrimeric alpha s, indicated that cytosolic alpha s is monomeric. These data indicate that alpha s is not exclusively localized to the plasma membrane and that agonist treatment redistributes this protein within target cells.
Evolution of immunoglobulin genes: VH families in the amphibian Xenopus.
Authors: Hsu E , Schwager J , Alt F W
Published: May 2006

We have isolated multiple independent cDNA clones that represent mRNA sequences of immunoglobulin heavy chains from the spleen of adult Xenopus laevis. These cDNA clones contained constant (C) region sequences that were either Cmu or a separate C region sequence believed to be Cnu. In individual cDNA clones the C region sequences were associated with independent heavy-chain variable region (VH) sequences that were classifiable into five distinct families. Genomic Southern blotting analyses with family-specific probes indicated that the haploid genome contains a minimum of 80 VH gene segments, a number similar to that found in various mammalian species. Multiple JH and putative DH segments were also identified (J, joining; D, diversity). Analyses of 13 independent VHDJH junctions suggest that combinatorial and junctional diversification mechanisms probably arose early in vertebrate evolution. Finally, comparison of Xenopus VH sequences to those from other vertebrates indicated conservation of V region framework residues that are responsible for the tertiary structure of the Fv throughout evolution.
Molecular cloning and sequencing of a cDNA of rat dopa decarboxylase: partial amino acid homologies with other enzymes synthesizing catecholamines.
Authors: Tanaka T , Horio Y , Taketoshi M , Imamura I , Ando-Yamamoto M , Kangawa K , Matsuo H , Kuroda M , Wada H
Published: May 2006

Dopa decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; aromatic-L-amino-acid carboxylase, EC 4.1.1.28) was purified from rat liver and its partial sequence was determined. Synthetic oligonucleotides were used to construct and screen rat liver cDNA libraries, and three clones were isolated and sequenced. The 2 kilobases of DDC cDNA cloned consisted of a 5'-noncoding segment of 78 nucleotides, a coding region of 1440 nucleotides, and a 3'-noncoding region of 438 nucleotides. The encoded protein of 480 amino acid residues had a molecular weight of 54,000. A special feature of the primary structure of rat DDC was a repeating structure consisting of 29 amino acid residues. A sequence of 58 amino acid residues, including this repeating structure of rat DDC, was found to show homologies with those of rat tyrosine hydroxylase, human dopamine beta-hydroxylase, and bovine phenylethanolamine N-methyltransferase, other mammalian enzymes that synthesize catecholamines. These results indicate that catecholamine biosynthetic enzymes are structurally related and suggest that their homologous domains are important for catechol-protein interactions.
Hydrophobic packing in T4 lysozyme probed by cavity-filling mutants.
Authors: Karpusas M , Baase W A , Matsumura M , Matthews B W
Published: May 2006

To probe the nature of the hydrophobic cores of proteins and to test potential ways of increasing protein thermostability, an attempt was made to improve the packing within T4 bacteriophage lysozyme by engineered amino acid replacements. Two mutations, Leu-133----Phe and Ala-129----Val, which were designed to fill the largest cavities that exist in the folded structure of the native protein, were constructed. The mutant proteins have normal activities and their thermal stabilities are marginally lower than that of wild-type lysozyme. Crystal structure analysis of the mutant proteins shows that the introduced amino acids are accommodated with very little perturbation of the three-dimensional structure. Incorporation of the more bulky hydrophobic residues within the core of the protein is expected to provide an increase in hydrophobic stabilization, but this is seen to be offset by the introduction of strain. Inspection of the mutant structures shows that in each case the introduced amino acid side chain is forced to adopt a non-optimal dihedral angle X1. Strain is also observed in the form of bond angle distortion and in unfavorable van der Waals contacts. The results illustrate how the observed core structures of proteins represent a compromise between the hydrophobic effect, which will tend to maximize the core packing density, and the strain energy that would be incurred in eliminating all packing defects. The results also suggest that mutations designed to increase protein stability by filling existing cavities may be effective in some cases but are unlikely to provide a general method for increasing protein stability.
Isoproterenol stimulates phosphorylation of the insulin-regulatable glucose transporter in rat adipocytes.
Authors: James D E , Hiken J , Lawrence J C
Published: May 2006

We have examined the acute effects of insulin and isoproterenol on the phosphorylation state of the insulin-regulatable glucose transporter (IRGT) in rat adipocytes. The IRGT was immunoprecipitated from either detergent-solubilized whole-cell homogenates or subcellular fractions of 32P-labeled fat cells and subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The 32P-labeled IRGT was detected by autoradiography as a species of apparent Mr 46,000. Insulin stimulated translocation of the IRGT from low-density microsomes to the plasma membrane but did not affect phosphorylation of the transporter in either fraction. Isoproterenol inhibited insulin-stimulated glucose transport by 40% but was without effect on the subcellular distribution of the transporter in either the presence or absence of insulin. Isoproterenol stimulated phosphorylation of the IRGT 2-fold. Incubating cells with dibutyryl-cAMP and 8-bromo-cAMP also stimulated phosphorylation 2-fold, and the transporter was phosphorylated in vitro when IRGT-enriched vesicles were incubated with cAMP-dependent protein kinase and [gamma-32P]ATP. These results suggest that isoproterenol stimulates phosphorylation of the IRGT via a cAMP-dependent pathway and that phosphorylation of the transporter may modulate its ability to transport glucose.
Transcriptional regulatory regions of testis-specific PGK2 defined in transgenic mice.
Authors: Robinson M O , McCarrey J R , Simon M I
Published: May 2006

The gene encoding testis-specific phosphoglycerate kinase 2 (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) is expressed only in meiotic and haploid male germ cells. Transgenic mice containing an 8-kilobase human genomic PGK2 gene express the human gene in a tissue-specific and developmentally regulated manner. To determine the nature and location of sequences controlling this expression, transgenic mice with various lengths of the human PGK2 5' region fused to the chloramphenicol acetyltransferase (CAT) gene were analyzed for expression. A 323-base-pair region 5' to the coding region was found to contain information essential for both tissue-specific and developmentally regulated expression of the CAT reporter gene. Transgenic mice containing a PGK2/luciferase-coding construct were compared with mice containing an equivalent CAT construct. Luciferase gene expression was also testis-specific and was more sensitive than CAT gene expression, but otherwise regulation of the two reporter genes was similar in the germ cells of transgenic mice. Translation of both PGK2/CAT and PGK2/luciferase fusion genes was seen concurrently with the first detectable transcripts.
Definition of a population of CD4-8- T cells that express the alpha beta T-cell receptor and respond to interleukins 2, 3, and 4.
Authors: Londei M , Verhoef A , De Berardinis P , Kissonerghis M , Grubeck-Loebenstein B , Feldmann M
Published: May 2006

Whereas most T cells express surface CD4 or CD8 molecules, a minority lacks both. CD4-8- cells usually express the gamma delta T-cell receptor, but here we describe a population of CD4-8- T cells from the peripheral blood that express the alpha beta heterodimer. These cells have different surface antigens than gamma delta+ T cells, expressing CD5 but lacking CD16, and differ in function from gamma delta+ T cells. CD4-8- alpha beta+ cells lack non-major histocompatibility complex-restricted cytolytic function but can be induced to lyse their target cells after activation of their T-cell receptors. A peculiar characteristic of these cells is their responsiveness to interleukin 3. Since these cells have not altered their phenotype or function over a 12-month period in culture, they appear to be mature T cells. The results indicate that normal human peripheral blood contains two subsets of CD4-8- T cells, expressing either gamma delta or alpha beta receptors, that differ in function, phenotype, and growth control.
Subunits of purified calcium channels: a 212-kDa form of alpha 1 and partial amino acid sequence of a phosphorylation site of an independent beta subunit.
Authors: De Jongh K S , Merrick D K , Catterall W A
Published: May 2006

Antibodies prepared against peptides CP2, CP4, and CP5, which occur within the first 1522 amino acid residues of the alpha 1 subunit of dihydropyridine-sensitive skeletal muscle calcium channels, specifically recognized a 175-kDa form of the alpha 1 subunit in immunoblots and immunoprecipitation experiments. In contrast, antibodies prepared against peptide CP1, which represents the C-terminal 18 amino acid residues predicted by cloning and sequence analysis of the alpha 1 subunit, recognized a minor, previously undescribed 212-kDa protein, which is the size predicted for the full length of the alpha 1 subunit from cDNA cloning [Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1987) Nature (London) 328, 313-318]. Both the 175-kDa and 212-kDa forms were phosphorylated by cAMP-dependent protein kinase and both were present in isolated transverse tubule membranes. The 175-kDa form may arise from posttranslational proteolytic cleavage of the C terminus of the 212-kDa form of the alpha 1 subunit predicted by cDNA cloning and sequence analysis. Partial amino acid sequencing of the 54-kDa beta subunit of the calcium channel indicated this protein was not derived from the proteolytically cleaved C terminus of the alpha 1 subunit. This analysis identified a threonine residue in the sequence (Lys/Arg)-Arg-Pro-Thr-Pro of the beta subunit that was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of this residue in the beta subunit may play a role in modulation of calcium channel function. Separate functional roles of the 175-kDa form of the alpha 1 subunit in excitation-contraction coupling and of the 212-kDa form in ion conductance are proposed.
Molecular analysis of O6-substituted guanine-induced mutagenesis of ras oncogenes.
Authors: Mitra G , Pauly G T , Kumar R , Pei G K , Hughes S H , Moschel R C , Barbacid M
Published: May 2006

We have designed an Ha-ras/thymidine kinase (TK) cassette that permits the incorporation of chemically synthesized adducts within specific domains of the rat Ha-ras protooncogene. This cassette has been used to evaluate the mutagenicity of O6-substituted guanine residues, including O6-methylguanine and O6-benzylguanine, incorporated within the 12th codon of this locus. Mutations were monitored by the ability of these modified Ha-ras DNAs to transform Rat4 TK-cells. Our results indicate that both types of O6-substituted guanines are substantially mutagenic, although the methyl substituent induced a 2-fold higher percentage of transformed Rat4 TK+ colonies than its bulkier benzyl analogue. Interestingly, the mutagenicity of both O6-substituted guanines was found to be independent of their relative position within codon 12, therefore suggesting that the specific activation of Ha-ras oncogenes by GGA----GAA mutations in tumors induced by methylating carcinogens might be due to differences in the accessibility of these guanine residues to the carcinogen rather than to a differential rate of repair. Molecular analysis of the mutations induced by these O6-substituted guanines indicated that O6-methylguanine exclusively induced G----A transitions. In contrast, O6-benzylguanine produced G----C and G----T transversions in addition to G----A transitions. These results suggest that O6-methylguanine and its bulkier analogue O6-benzylguanine may induce mutagenesis by different mechanisms.
Effects of cholesterol or gramicidin on slow and fast motions of phospholipids in oriented bilayers.
Authors: Peng Z Y , Simplaceanu V , Dowd S R , Ho C
Published: May 2006

Nuclear spin-lattice relaxation both in the rotating frame and in the laboratory frame is used to investigate the slow and fast molecular motions of phospholipids in oriented bilayers in the liquid crystalline phase. The bilayers are prepared from a perdeuterated phospholipid labeled with a pair of 19F atoms at the 7 position of the 2-sn acyl chain. Phospholipid-cholesterol or phospholipid-gramicidin interactions are characterized by measuring the relaxation rates as a function of the bilayer orientation, the locking field, and the temperature. Our studies show that cholesterol or gramicidin can specifically enhance the relaxation due to slow motions in phospholipid bilayers with correlation times tau s longer than 10(-8) sec. The perturbations of the geometry of the slow motions induced by cholesterol are qualitatively different from those induced by gramicidin. In contrast, the presence of cholesterol or gramicidin slightly suppresses the fast motions with correlation times tau f = 10(-9) to 10(-10) sec without significantly affecting their geometry. Weak locking-field and temperature dependences are observed for both pure lipid bilayers and bilayers containing either cholesterol or gramicidin, suggesting that the motions of phospholipid acyl chains may have dispersed correlation times.
Translocation of the FGR protein-tyrosine kinase as a consequence of neutrophil activation.
Authors: Gutkind J S , Robbins K C
Published: May 2006

Recent studies of the FGR protooncogene have shown that expression of its mRNA is limited to mature peripheral blood granulocytes, monocytes, and tissue macrophages. In the present study, we have investigated p55c-fgr expression in normal human neutrophils [polymorphonuclear leukocytes (PMN)] and have found enzymatically active p55c-fgr to be abundant in lysates of PMN and murine fibroblasts transfected with a FGR expression plasmid but not control cells. Fractionation studies revealed that neutrophil p55c-fgr was present in plasma membrane-enriched fractions as well as fractions containing secondary and tertiary granules. Little change in the distribution of p55c-fgr or FGR kinase activity was observed under conditions favoring tertiary granule release. In contrast, when secondary granule secretion was induced with the chemoattractant peptide, formyl-Met-Leu-Phe, a marked decrease in p55c-fgr and FGR kinase was observed in fractions depleted of secondary granules. Concomitantly, the relative concentration of p55c-fgr and its enzymatic activity were increased in fractions containing plasma membrane. From these findings we conclude that p55c-fgr is associated with functional secretory granules and is redistributed within normal neutrophils in response to their activation.
Structural dependence of the inhibition of mitochondrial respiration and of NADH oxidase by 1-methyl-4-phenylpyridinium (MPP+) analogs and their energized accumulation by mitochondria.
Authors: Ramsay R R , Youngster S K , Nicklas W J , McKeown K A , Jin Y Z , Heikkila R E , Singer T P
Published: May 2006

Nineteen structural analogs of 1-methyl-4-phenylpyridinium (MPP+) were studied for their capacity to inhibit the mitochondrial oxidation of NAD+-linked substrates and the aerobic oxidation of NADH in inner membrane preparations from cardiac mitochondria. In the majority of cases, a good correlation was found between the two inhibition effects monitored. A few compounds were effective inhibitors of NADH oxidase but had only marginal effects on mitochondrial respiration. From studies of their accumulation by mitochondria, it appears likely that the latter compounds are not effectively concentrated by intact mitochondria by the electrical gradient and, in part for this reason, cannot reach sufficiently high concentrations at the appropriate binding site of NADH dehydrogenase. In addition, evidence is presented that the penetration of pyridinium analogs to the inhibition site in the NADH dehydrogenase complex may also be rate limiting. The data support the thesis that, for a substituted tetrahydropyridine to be acutely neurotoxic, its pyridinium oxidation product must be actively accumulated in the mitochondria and must inhibit NADH-ubiquinone oxidoreductase in its membrane environment.
Structure and developmental expression of a sea urchin fibrillar collagen gene.
Authors: D'Alessio M , Ramirez F , Suzuki H R , Solursh M , Gambino R
Published: May 2006

We have isolated and characterized cDNA and genomic clones that specify a Paracentrotus lividus procollagen chain. The cDNAs code for 160 uninterrupted Gly-Xaa-Yaa triplets and a 252-amino acid carboxyl propeptide. Analysis of the deduced amino acid sequences indicated that the sea urchin polypeptide exhibits structural features that are characteristic of the fibril-forming class of collagen molecules. Partial characterization of two genomic recombinants revealed that the 3' end of the echinoid gene displays a complex organization that closely resembles that of a prototypical vertebrate fibrillar collagen gene. In situ and Northern (RNA) blot hybridizations established the size, time of appearance, and tissue distribution of the collagen transcripts in the developing sea urchin embryo. Collagen mRNA, approximately equal to 6 kilobases in size, is first detected in the forming primary mesenchyme cells of late blastulae where it progressively accumulates until the free swimming/feeding pluteus larval stage. Interestingly, collagen transcripts are also detected in the forming secondary mesenchyme cells of late gastrulae, and by the prism stage, their derivatives appear to be the most intensively labeled cells.
Histone H1 in two subspecies of Chironomus thummi with different genome sizes: homologous chromosome sites differ largely in their content of a specific H1 variant.
Authors: Mohr E , Trieschmann L , Grossbach U
Published: May 2006

Chromatin of Chironomus thummi (Diptera) contains seven sequence variants of histone H1. A structurally divergent H1, variant I-1, accounts for about 20% of the total H1 in C. th. piger and for about 30% in C. th. thummi. Monoclonal antibodies against this protein have been induced and have revealed its restriction to the centromeres and to a limited number of other bands in the salivary gland chromosomes. Indirect immunofluorescence of the somatically paired homologous chromosomes of F1 hybrids indicates that the difference between the two subspecies in H1 I-1 content largely depends on differences situated at a number of distinct homologous chromosome bands. These bands were intensely decorated by antibodies against H1 I-1 in C. th. thummi but appeared virtually black in C. th. piger. The same bands, however, were decorated equally in both subspecies by an antibody that reacts with other H1 variants but does not recognize H1 I-1 and by a polyclonal anti-H1 antibody. The results suggest that H1 variant I-1 is characteristic of a specific type of chromatin that is confined to distinct chromosome segments and that is more frequent in the subspecies C. th. thummi, which has a 27% larger genome.
Lipoprotein binding and endosomal itinerary of the low density lipoprotein receptor-related protein in rat liver.
Authors: Lund H , Takahashi K , Hamilton R L , Havel R J
Published: May 2006

The high affinity of 45Ca binding to the low density lipoprotein receptor (LDL-R) and the LDL-R-related protein (LRP) was utilized to study the subcellular distribution of these two proteins in rat liver. Like the LDL-R, LRP was manyfold enriched in rat liver endosomal membranes with a relative distribution in early and late endosomal compartments consistent with recycling between endosomes and the cell surface. The high concentration of LRP in hepatic endosomal membranes greatly facilitated demonstration of Ca-dependent binding of apolipoprotein E- and B-containing lipoproteins in ligand blots. LRP was severalfold more abundant than the LDL-R in hepatic parenchymal cells, showed extensive degradation in hepatic endosomes, and was found in high concentrations in the Golgi apparatus and endoplasmic reticulum. These data suggest a high rate of synthesis of LRP that appeared to be unaffected by treatment of rats with estradiol. The repeating cysteine-rich A-motif found in the ligand-binding domain of LRP appeared to be responsible for Ca binding by LRP, LDL-R, and complement factor C9 and accounted for immunological cross-reactivity among these proteins. Weaker ligand-blotting properties and an extraordinary susceptibility to proteolysis most likely contribute to the difficulty of detecting LRP in conventional assays for lipoprotein receptors. Our data suggest an extensive proteolytic processing of this protein and are consistent with a functional role of LRP in lipoprotein metabolism.
Inositol trisphosphate and diacylglycerol can differentially modulate gene expression in Dictyostelium.
Authors: Ginsburg G , Kimmel A R
Published: May 2006

We have previously shown that several genes expressed during Dictyostelium development could be induced in shaking culture by exogenous cAMP, even though the accumulation of intracellular cAMP was inhibited. The use of selected cAMP analogs indicated that the exogenous cAMP functioned by activating the cell surface cAMP receptor and not by interacting with the regulatory subunit of the intracellular cAMP-dependent protein kinase. Although some genes in Dictyostelium appear to be regulated by intracellular cAMP, these data suggest that this is not the case for all genes regulated by cAMP. Intracellular second messengers other than cAMP may, therefore, promote the expression of these other genes. Here, we have examined inositol trisphosphate and diacylglycerol as candidates for such mediators of signal transduction. We have studied three genes that exhibit disparate modes of temporal and spatial expression during development of Dictyostelium. In shaking cultures, maximal levels of expression of each are dependent on the accumulation of or exposure to extracellular cAMP. We show that the addition of inositol trisphosphate and/or diacylglycerol to cells in shaking culture has distinct effects on the expression of each gene and, under specific conditions, can bypass the requirement for extracellular cAMP. These data suggest that extracellular cAMP interacting with its cell surface receptor may promote synthesis of inositol trisphosphate and diacylglycerol to regulate gene expression and aspects of differentiation in Dictyostelium.