bluuebunny/crossref_metadata_2025 huggingface dataset:
Three-dimensional structure of cyanomet-sulfmyoglobin C.
Authors: Evans S V , Sishta B P , Mauk A G , Brayer G D
Published: May 2006

The atomic structure of horse heart cyanomet-sulfmyoglobin C has been established by x-ray crystallographic techniques to a resolution of 2.0 A with an R value of 0.129. The protoheme IX prosthetic group of this thermodynamically stable sulfmyoglobin derivative has been converted to a chlorin in which the pyrrole ring bearing the 4-vinyl group is saturated and possesses an exocyclic thiolene ring. This study provides the three-dimensional structure of a protein with an iron-chlorin prosthetic group. The overall conformation of the surrounding polypeptide chain of the modified protein is very similar to that of the native protein. However, the addition of the sulfur atom has caused a distortion of the prosthetic group from that in the native protein to result in the repositioning of the side chains of some residues in the heme pocket.
Developmental changes in the expression and compartmentalization of the glucocorticoid receptor in embryonic retina.
Authors: Gorovits R , Ben-Dror I , Fox L E , Westphal H M , Vardimon L
Published: May 2006

Inducibility by glucocorticoids of the glutamine synthetase gene in chicken embryo retina and the transcriptional activity of the glucocorticoid receptor (GR) greatly increase between embryonic days 6 and 10 (E6, E10), although the level of GR does not markedly change during that time. This apparent discrepancy was investigated by examining the pattern of GR expression in undifferentiated E6 retina and in E10 retina, which consists mostly of differentiated cells. Two GR isoforms, 90 and 95 kDa, were found to be expressed at both of these ages at a similar total level but in different proportions: in E6 retina the level of the 90-kDa isoform was higher, whereas in E10 retina the 95-kDa receptor was higher. However, following treatment of the retinas with cortisol, the 95-kDa isoform became the predominant receptor at both ages. Immunohistochemical analysis revealed that the cellular localization of GR markedly changed in the course of development: in the undifferentiated E6 retina GR was expressed in virtually all cells, whereas in the more differentiated E10 and E12 retina, GR was detected only in Müller glia cells. The latter represent approximately 20% of the cells in this tissue and are the only cells in which glucocorticoid hormone induces the glutamine synthetase gene. We suggest that the compartmentalization of GR in Müller glia is a major aspect of the mechanism that modulates receptor activity during retina development and results in the temporal increase in the inducibility of glutamine synthetase and its specific localization in Müller glia cells.
X-ray structure determination at 2.6-A resolution of a lipoate-containing protein: the H-protein of the glycine decarboxylase complex from pea leaves.
Authors: Pares S , Cohen-Addad C , Sieker L , Neuburger M , Douce R
Published: May 2006

H-protein, a lipoic acid-containing protein of the glycine decarboxylase (EC 1.4.4.2) complex from pea (Pisum sativum) was crystallized from ammonium sulfate solution at pH 5.2 in space group P3(1)21. The x-ray crystal structure was determined to 2.6-A resolution by multiple isomorphous replacement techniques. The structure was refined to an R value of 23% for reflections between 15- and 2.6-A resolution (F > 2 sigma), including the lipoate moiety and 50 water molecules, for the two protein molecules of the asymmetric unit. The 131-amino acid residues form seven beta-strands arranged into two antiparallel beta-sheets forming a "sandwich" structure. One alpha-helix is observed at the C-terminal end. The lipoate cofactor attached to Lys-63 is located in the loop of a hairpin configuration. The lipoate moiety points toward the residues His-34 and Asp-128 and is situated at the surface of the H-protein. This allows the flexibility of the lipoate arm. This is the first x-ray determination of a lipoic acid-containing protein, and the present results are in agreement with previous theoretical predictions and NMR studies of the catalytic domains of lipoic acid- and biotin-containing proteins.
An algorithm to generate low-resolution protein tertiary structures from knowledge of secondary structure.
Authors: Monge A , Friesner R A , Honig B
Published: May 2006

An algorithm is described to assemble the three-dimensional fold of a protein starting from its secondary structure. A reduced representation of the polypeptide chain is used together with a crude potential based on pair hydrophobicities. The method is shown to be successful in locating the native topology for two 4-alpha-helix bundles, myohemerythrin and cytochrome b-562.
In vivo production of human factor VII in mice after intrasplenic implantation of primary fibroblasts transfected by receptor-mediated, adenovirus-augmented gene delivery.
Authors: Zatloukal K , Cotten M , Berger M , Schmidt W , Wagner E , Birnstiel M L
Published: May 2006

Hemophilia A is caused by defects in the factor VIII gene. This results in life-threatening hemorrhages and severe arthropathies. Today, hemophiliacs are treated with human blood-derived factor VIII. In the future, it may be possible to use gene therapy to avoid long-term complications of conventional therapy and to improve the quality of life. However, initial gene therapy models using retroviral vectors and nonviral gene transfer techniques to introduce factor VIII gene constructs have been hampered by low expression levels of factor VIII. We show here that high expression levels of the B-domain-deleted human factor VIII in primary mouse fibroblasts and myoblasts are obtained by using receptor-mediated, adenovirus-augmented gene delivery (transferrinfection). We demonstrate that, presumably owing to the high molecular weight of factor VIII or its metabolic instability, secretion into the blood and attainment of therapeutic in vivo levels of factor VIII is achieved only if transfected autologous primary fibroblasts or myoblasts are delivered to the liver or spleen, but not if myoblasts are implanted into muscle, a strategy known to be successful for factor IX delivery.
Activation of the pp90rsk and mitogen-activated serine/threonine protein kinases by ionizing radiation.
Authors: Kharbanda S , Saleem A , Shafman T , Emoto Y , Weichselbaum R , Kufe D
Published: May 2006

The cellular response to ionizing radiation (IR) includes induction of the c-jun and EGR1 early response genes. The present work has examined potential cytoplasmic signaling cascades that transduce IR-induced signals to the nucleus. The results demonstrate activation of the 40S ribosomal protein S6 kinase, pp90rsk, in human U-937 myeloid leukemia cells. Partial purification of pp90rsk by affinity chromatography demonstrated an increase in S6 peptide phosphorylation when comparing irradiated with control cells. IR-induced activation of pp90rsk was further confirmed in immune-complex kinase assays. In contrast to these findings, there was no detectable induction of pp70S6K. Previous work has demonstrated that mitogen-activated protein kinase activates pp90rsk. The present results further show that IR treatment is associated with induction of mitogen-activated protein kinase activity and that this event is temporally related to activation of pp90rsk and early response gene expression. These findings suggest that activation of the mitogen-activated protein kinase/pp90rsk cascade is involved in the response of cells to IR exposure.
GTP-dependent polymerization of Escherichia coli FtsZ protein to form tubules.
Authors: Bramhill D , Thompson C M
Published: May 2006

The FtsZ protein is a GTPase that is essential for cell division in Escherichia coli. During cytokinesis, FtsZ localizes to a ring at the leading edge of septum synthesis. We report the GTP-dependent polymerization of purified FtsZ measured by sedimentation and light scattering. Electron microscopy of polymerized FtsZ revealed structures including tubules 14-20 nm in diameter with longitudinal arrays of protofilaments. FtsZ depolymerized upon removal of GTP and repolymerized after subsequent GTP addition. Mutant FtsZ84 protein polymerized inefficiently, suggesting that polymerization is important for the cellular role of FtsZ in division. The possibility that tubules of FtsZ protein form a cytoskeleton involved in septum synthesis is consistent with our data.
A small nuclear GTP-binding protein from tomato suppresses a Schizosaccharomyces pombe cell-cycle mutant.
Authors: Ach R A , Gruissem W
Published: May 2006

Ran is a 25-kDa Ras-related nuclear GTP-binding protein which is very highly conserved in humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Ran has been found to form a stable, noncovalent complex with the chromatin-associated protein RCC1, a negative regulator of mitosis. In Sch. pombe, a temperature-sensitive mutation in the RCC1 homolog encoded by the pim1 gene causes premature induction of mitosis, and this mutation can be suppressed by overexpression of the Ran homolog encoded by spi1. We report here the cloning of three Ran cDNAs from tomato. The Ran protein is very highly conserved among plants, animals, and fungi. In tomato, Ran mRNA is expressed in all tissues examined, even those with little or no cell division, indicating that Ran in plants may have functions other than just control of mitosis. We have found that the tomato Ran protein can direct a beta-glucuronidase reporter protein to the plant cell nucleus, confirming that Ran is a nuclear protein in plants. We show that the tomato Ran protein can suppress the Sch. pombe pim1 mutation, indicating that the tomato Ran protein and the Sch. pombe spi1 protein are functionally homologous.
Tau-beta-galactosidase, an axon-targeted fusion protein.
Authors: Callahan C A , Thomas J B
Published: May 2006

The most commonly used enzymatic reporter molecule, Escherichia coli beta-galactosidase (beta-gal; beta-D-galactoside galactohydrolase, EC 3.2.1.23), fails to readily diffuse into axons; consequently, the morphologies of beta-gal-labeled neurons cannot directly be determined. For analysis of neuronal pathfinding and synaptic connectivity, this information is essential. We have constructed an axon-targeted beta-gal reporter by fusing the cDNA encoding the bovine microtubule-binding protein, tau, to lacZ, the E. coli gene encoding beta-gal. This reporter labels cell bodies and axons when expressed by developing and adult Drosophila neurons. It also reveals the entire cellular extent of nonneuronal cells such as muscle fibers and glia. To generate neuronal markers for studies of Drosophila neural development, we constructed a tau-beta-gal enhancer-trap transposon. From 1500 independent lines generated by mobilization of this transposon, we have isolated a set of useful markers for specific subsets of neurons, glia, and muscles. Since the tau cDNA-lacZ reporter utilizes bovine tau, it may also effectively target beta-gal in vertebrate neurons and prove to be a useful reagent for the analysis of vertebrate nervous systems.
Hydrolysis and transesterification of platelet-activating factor by lecithin-cholesterol acyltransferase.
Authors: Liu M , Subbaiah P V
Published: May 2006

Purified lecithin-cholesterol acyltransferase (LCAT, EC 2.3.1.43) from human plasma was found to hydrolyze platelet-activating factor (PAF) to lyso-PAF and acetate. In addition, it catalyzed the transfer of the acetate group from PAF to lysophosphatidylcholine, forming lyso-PAF and a 1-acyl analog of PAF. In contrast to the cholesterol-esterification reaction carried out by the enzyme, the hydrolysis and transacetylation of PAF by LCAT did not require an apoprotein activator and were not inhibited by sulfhydryl inhibitors but were inhibited by serum albumin. When added to a proteoliposome substrate of LCAT or to whole plasma, PAF inhibited cholesterol esterification by LCAT competitively. PAF acetylhydrolase (EC 3.1.1.47), purified from human plasma, also catalyzed the transfer of acetate from PAF to lysophosphatidylcholine. However, the LCAT-catalyzed reactions of PAF were not due to contamination with PAF acetylhydrolase, since the ratio of acetyl transfer to acetyl hydrolysis was 3 times greater for LCAT, when compared with PAF acetylhydrolase under identical conditions. Furthermore, recombinant human LCAT secreted by baby hamster kidney cells also catalyzed the hydrolysis and transacetylation of PAF. These results demonstrate that LCAT can inactivate PAF in plasma by transacetylation and suggest that it may have a role in the metabolism of PAF, and possibly of oxidized phospholipids, in plasma.
Na(+)-, ouabain-, Ca(2+)-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump alpha subunits.
Authors: Ishii T , Lemas M V , Takeyasu K
Published: May 2006

Using the chicken sarcoplasmic/endoplasmic reticulum Ca2+ (SERCA)-ATPase as a parental molecule and replacing various portions with the corresponding portions of the chicken Na+,K(+)-ATPase alpha 1 subunit, Ca2+/thapsigargin- and Na+/ouabain-sensitive domains critical for these P-type ATPase activities were identified. In the chimera, [n/c]CC, the amino-terminal amino acids Met-1 to Asp-162 of the SERCA (isoform 1) (SERCA1) ATPase were replaced with the corresponding portion (Met-1-Asp-200) of the Na+,K(+)-ATPase alpha 1 subunit. In the chimera CC[c/n], the carboxyl-terminal amino acids (Ser-830 to COOH) of the SERCA1 ATPase were replaced with the corresponding segment (Leu-861 to COOH) of the Na+,K(+)-ATPase alpha 1 subunit, and in the chimera CNC, the middle part (Gly-354-Lys-712) of the SERCA1 ATPase was exchanged with the Na+,K(+)-ATPase alpha 1 subunit (Gly-378-Lys-724). None of the chimeric molecules exhibited any detectable ouabain-sensitive Na+,K(+)-ATPase activity, but they did exhibit thapsigargin-sensitive Ca(2+)-ATPase activity. Therefore, the segments Ile-163-Gly-354 and Lys-712-Ser-830 of the SERCA1 ATPase are sufficient for Ca2+ and thapsigargin sensitivity. The SERCA1-ATPase activity of [n/c]CC, but not of CCC, CNC, or CC[c/n], was further stimulated by addition of Na+ in the assay medium containing Ca2+. This additional stimulation of SERCA1-ATPase activity by Na+ was abolished when the amino-terminal region (Met-1-Leu-69) of [n/c]CC was deleted ([delta n/c]CC). In the absence of Na+, the SERCA1-ATPase activity of [n/c]CC was inhibited by ouabain, and, in the presence of Na+, its activity was stimulated by this drug. On the other hand, the ATPase activity of [delta n/c]CC was not affected by ouabain, although [delta n/c]CC can still bind [3H]ouabain. These results suggest that a distinct Na(+)-sensitive domain (Na+ sensor) located within the restricted amino-terminal region (Met-1-Leu-69) of the Na+,K(+)-ATPase alpha 1 subunit regulates ATPase activity. The Na+ sensor also controls ouabain action in concert with the major ouabain-binding region between Ala-70 and Asp-200 of alpha 1 subunit.
Preimplantation single-cell analysis of multiple genetic loci by whole-genome amplification.
Authors: Snabes M C , Chong S S , Subramanian S B , Kristjansson K , DiSepio D , Hughes M R
Published: May 2006

Due to the limited amount of DNA in a single diploid cell, preimplantation genetic diagnosis has relied on single- or dual-locus analyses in biopsied blastomers. We have applied single-cell whole-genome preamplification to PCR-based analysis of multiple disease loci from the same diploid cell. This method allows diagnosis of multiple disease genes, analysis of multiple exons/introns within a gene, or corroborative embryo-sex assignment and specific mutation detection at sex-linked loci. A blinded study of six genetic loci was performed with whole-genome preamplification followed by nested PCR. Amplification was observed in 103 of 105 assays (98%) and a correct diagnosis was made in 98%. All human blastomeres were correctly diagnosed (100%) at loci where the genotype could be confirmed, attesting to the reliability of the technique. Preamplification has now been applied successfully to the analysis of the two major mutations responsible for Tay-Sachs disease and of a common restriction polymorphism in the gene responsible for hemophilia A. The fidelity and length of product derived from this preamplification step make it an appealing technique for preimplantation genetic diagnoses requiring analyses at more than one locus.
The alpha 3 chain of type IV collagen induces autoimmune Goodpasture syndrome.
Authors: Kalluri R , Gattone V H , Noelken M E , Hudson B G
Published: May 2006

Human Goodpasture syndrome is a lethal form of autoimmune disease that is characterized by pulmonary hemorrhage and glomerulonephritis. The tissue injury is mediated by autoantibodies that bind to glomerular and alveolar basement membrane. The target autoantigen is alpha 3(IV) collagen, one of six genetically distinct chains that comprise type IV collagen, and the epitope is sublocalized to the noncollagenous domain (NC1) of the alpha 3 chain. The present study reports the unique capacity of alpha 3(IV)NC1 dimer from bovine kidney to aberrantly engage the immune system of rabbits to respond to self, mimicking the organ-specific form of the human disease, whereas the other chains of type IV collagen are nonpathogenic. However, alpha 3(IV)NC1 hexamer was nonpathogenic, suggesting the exposure of a pathogenic epitope upon dissociation of hexamer into dimers. Exposure of the pathogenic epitope by infection or organic solvents, events which are thought to precede Goodpasture syndrome, may be the principal factor in the etiology of the disease. The pathogenicity of alpha 3(IV) collagen brings full circle a decade of research that has identified four novel chains (alpha 3-alpha 6) of type IV collagen.
Cyclic nucleotide- and inositol phosphate-gated ion channels in lobster olfactory receptor neurons.
Authors: Hatt H , Ache B W
Published: May 2006

The idea of having two second messenger pathways in olfaction, one mediated by cAMP and the other by inositol 1,4,5-trisphosphate, is supported by evidence that both second messengers directly activate distinct ion channels in the outer dendrite of lobster olfactory receptor neurons. Evidence that both types of second messenger-gated channels can occur in the same patch of membrane suggests that channels of both types can be expressed in one neuron. Evidence of more than one type of inositol phosphate-gated channel in this highly specialized region of the neuron furthers the idea that the output of individual olfactory receptor cells is regulated through multiple effectors and allows that effector diversity may contribute to functional diversity among olfactory receptor cells.
Partial rescue of a lethal phenotype of fragile bones in transgenic mice with a chimeric antisense gene directed against a mutated collagen gene.
Authors: Khillan J S , Li S W , Prockop D J
Published: May 2006

Previously, transgenic mice were prepared that developed a lethal phenotype of fragile bones because they expressed an internally deleted mini-gene for the pro alpha 1(I) chain of human type I procollagen. The shortened pro alpha 1(I) chains synthesized from the human transgene bound to and produced degradation of normal pro alpha 1(I) chains synthesized from the normal mouse alleles. Here we assembled an antisense gene that was similar to the internally deleted COL1A1 minigene but the 3' half of the gene was inverted so as to code for an antisense RNA. Transgenic mice expressing the antisense gene had a normal phenotype, apparently because the antisense gene contained human sequences instead of mouse sequences. Two lines of mice expressing the antisense gene were bred to two lines of transgenic mice expressing the mini-gene. In mice that inherited both genes, the incidence of the lethal fragile bone phenotype was reduced from 92% to 27%. The effects of the antisense gene were directly demonstrated by an increase in the ratio of normal mouse pro alpha 1(I) chains to human mini-pro alpha 1(I) chains in tissues from mice that inherited both genes and had a normal phenotype. The results raise the possibility that chimeric gene constructs that contain intron sequences and in which only the second half of a gene is inverted may be particularly effective as antisense genes.
The POU domain protein Tst-1 and papovaviral large tumor antigen function synergistically to stimulate glia-specific gene expression of JC virus.
Authors: Renner K , Leger H , Wegner M
Published: May 2006

Synergism between transcriptional activators is a powerful way of potentiating their function. Here we show that the glial POU domain protein Tst-1 (also known as Oct-6 and SCIP) and large tumor antigen (T antigen) synergistically increased transcription from both the early and the late promoters of papovavirus JC in glial cells. Synergism between both proteins did not require T-antigen-mediated DNA replication or direct binding of T antigen to the promoter. The ability of T antigen to functionally cooperate with Tst-1 was contained within its N-terminal region, shown by the fact that small tumor antigen (t antigen) could substitute for T antigen in transfection experiments. In addition to this functional synergism, a direct interaction between Tst-1 and T antigen was observed in vitro. Using deletion mutants of Tst-1 and T antigen, the POU domain of Tst-1 and the N-terminal region of T antigen were found to participate in this interaction. Because of the low levels of Tst-1 present in oligodendrocytes, synergism between Tst-1 and T antigen could be an important factor in establishing the lytic infection of oligodendrocytes by JC virus during the course of the fatal demyelinating disease progressive multifocal leukoencephalopathy.
Multiple pathways of copy control of gamma replicon of R6K: mechanisms both dependent on and independent of cooperativity of interaction of tau protein with DNA affect the copy number.
Authors: Miron A , Patel I , Bastia D
Published: May 2006

The ability of a replication initiator protein to promote intermolecular pairing of two replication origins resulting in the turning off of the origin pair has been called handcuffing. We have endeavored to test the validity of the handcuffing model by isolating two mutant forms of the tau initiator protein of R6K that elicit high copy number phenotype. We have discovered that one mutant called tau 113 yielded a 3.6-fold increase in copy number of a gamma replicon with a concomitant impairment of its ability to loop DNA and to pair binding sites (iterons) in comparison with normal tau, thus supporting the handcuffing model. A second mutant called tau 108, on the other hand, elicited a 3-fold increase in copy number without showing any measurable loss in its ability to loop and pair gamma iterons. Both mutant forms of the wild-type tau protein showed no detectable differences in their affinity of binding to the gamma iterons. Thus, the phenotype of tau 108 is consistent with the proposition that copy number control involves macromolecular interactions other than cooperativity at a distance of tau or interaction of tau with the primary binding sites at gamma. Taken together, the results are consistent with the notion that tau-mediated handcuffing is a mechanism, but not the only mechanism, of copy control in R6K. Interaction of tau with host proteins is likely to provide additional facets of the copy control mechanism.
Genetic variants of human serum albumin in Italy: point mutants and a carboxyl-terminal variant.
Authors: Madison J , Galliano M , Watkins S , Minchiotti L , Porta F , Rossi A , Putnam F W
Published: May 2006

Of the > 50 different genetic variants of human serum albumin (alloalbumins) that have been characterized by amino acid or DNA sequence analysis, almost half have been identified in Italy through a long-term electrophoretic survey of serum. Previously we have reported structural studies of 11 Italian alloalbumins with point mutations, 2 different carboxyl-terminal variants, and 1 case of analbuminemia in an Italian family. This article describes confirmation by DNA sequencing of mutations previously inferred from protein sequencing of 4 of the above alloalbumins; it also reports the mutations identified by protein and DNA sequence analysis of 4 other Italian alloalbumins not previously recorded: albumin Larino, His3-->Tyr; Tradate-2 (protein sequencing only), Lys225-->Gln; Caserta, Lys276-->Asn; and Bazzano, a carboxyl-terminal variant. The first 3 have point mutations that produce a single amino acid substitution, but a nucleotide deletion causes a frameshift and an altered and truncated carboxyl-terminal sequence in albumin Bazzano. In these 4 instances the expression of the alloalbumin is variable, ranging from 10% to 70% of the total albumiN, in contrast to the usual 50% each for the normal and mutant albumin. The distribution of point mutations in the albumin gene is nonrandom; most of the 47 reported point substitutions involve charged amino acid residues on the surface of the molecule that are not concerned with ligand-binding sites.
Positive selection of self- and alloreactive CD8+ T cells in Tap-1 mutant mice.
Authors: Aldrich C J , Ljunggren H G , Van Kaer L , Ashton-Rickardt P G , Tonegawa S , Forman J
Published: May 2006

Mice with a homozygous deletion in their Tap-1 gene (-/- mice) express very low levels of cell membrane major histocompatibility complex class I molecules and have < 1% peripheral CD8+ T cells. We show that these -/- mice but not their +/- littermates display strong primary syngeneic anti-H-2Kb and -Db-specific responses mediated by CD8+ T cells. These responses are augmented by in vivo priming. Further, -/- mice primed in vivo with H-2d alloantigens generate an anti-H-2d response which appears nearly as strong as that found in +/- littermates. Both -/- anti-H-2b and anti-H-2d T cells do not recognize target cells from Tap-1 -/- animals or Tap-2-deficient RMA-S cells. Thus, some CD8+ anti-self and alloreactive T cells can be selected in the absence of Tap proteins.
Wheat acetyl-coenzyme A carboxylase: cDNA and protein structure.
Authors: Gornicki P , Podkowinski J , Scappino L A , DiMaio J , Ward E , Haselkorn R
Published: May 2006

cDNA fragments encoding part of wheat (Triticum aestivum) acetyl-CoA carboxylase (ACC; EC 6.4.1.2) were cloned by PCR using primers based on the alignment of several biotin-dependent carboxylases. A set of overlapping clones encoding the entire wheat ACC was then isolated by using these fragments as probes. The cDNA sequence contains a 2257-amino acid reading frame encoding a 251-kDa polypeptide. The amino acid sequence of the most highly conserved domain, corresponding to the biotin carboxylases of prokaryotes, is 52-55% identical to ACC of yeast, rat, and diatom. Identity with the available C-terminal amino acid sequence of maize ACC is 66%. The biotin attachment site has the typical eukaryotic EVMKM sequence. The cDNA does not encode an obvious chloroplast targeting sequence. Various cDNA fragments hybridize in Northern blots to a 7.9-kb mRNA. Southern analysis with cDNA probes revealed multiple hybridizing fragments in hexaploid wheat DNA. Some of the wheat cDNA probes also hybridize with ACC-specific DNA from other plants, indicating significant conservation among plant ACCs.
Differentiation between binding sites for angiotensin II and nonpeptide antagonists on the angiotensin II type 1 receptors.
Authors: Schambye H T , Hjorth S A , Bergsma D J , Sathe G , Schwartz T W
Published: May 2006

To characterize binding sites for nonpeptide angiotensin antagonists on the human angiotensin II receptor type 1 (AT1 receptor) we have systematically exchanged segments of the human receptor with corresponding segments from a homologous Xenopus laevis receptor, which does not bind the nonpeptide compounds. Substitution of transmembrane segment VII of the human AT1 receptor dramatically reduced the binding affinity of all of the 11 nonpeptide antagonists tested (55- to > 2000-fold) with no effect on the binding of angiotensin. The affinity for the nonpeptide compounds decreased additionally one order of magnitude when transmembrane segment VI and the connecting extracellular loop 3 from the Xenopus receptor were also introduced into the human AT1 receptor. Exchanges of smaller segments and single residues in transmembrane segments VI and VII and extracellular loop 3 revealed that the binding of nonpeptide antagonists was dependent on nonconserved residues located deep within the transmembrane segments VI and VII, in particular Asn295 in transmembrane segment VII. Surprisingly, all exchanges in transmembrane segment VII, including the Asn295 to Ser substitution, had a more pronounced effect on the binding of the competitive antagonists relative to the insurmountable antagonists. It is concluded that the binding mode for peptide and nonpeptide ligands on the AT1 receptor is rather different and that competitive and insurmountable antagonists presumably bind to overlapping but distinct sites located in transmembrane segments VI and VII.
A direct mechanism for sensing low oxygen levels by central neurons.
Authors: Jiang C , Haddad G G
Published: May 2006

The cascade of cellular events that is triggered by low O2 levels in the central nervous system depends on initial sensing mechanisms that can be crucial in determining the overall cell response, adaptation, or injury. In this report, we demonstrate that the activity of an identified K+ channel is regulated directly by environmental O2. Membrane ionic currents were recorded from neurons of the neocortex and the substantia nigra and studied by using whole-cell or excised membrane patches. O2 deprivation reversibly induced an initial transient increase in whole-cell outward currents, and this was followed by a pronounced decrease in these currents. In cell-free excised membrane patches, lack of O2 reversibly inhibited a class of K+ channels that are inhibited by ATP and activated by Ca2+. K+ channel inhibition depended on pO2 level, with a 50% inhibition at approximately 11 torr (1 torr = 6.9 kPa). By the use of specific agents that chelate metal in metal-containing O2-sensing centers, including heme, nonheme iron, copper, and flavin, we also demonstrated that iron-center but not copper-center blockers inhibited the channel in excised patches in a similar fashion as low pO2. These results strongly suggest that K+ channel activity is modulated during O2 deprivation by nonheme iron-containing proteins that are associated with channel molecules, thus providing evidence for a direct O2-sensing mechanism in neuronal membranes.
Photosystem II reaction center damage and repair cycle: chloroplast acclimation strategy to irradiance stress.
Authors: Vasilikiotis C , Melis A
Published: May 2006

A daily occurrence in the life of a plant is the function of a photosystem II (PSII) damage and repair cycle in chloroplasts. This unique phenomenon involves the frequent turnover of D1, the 32-kDa reaction-center protein of PSII (chloroplast psbA gene product). In the model organism Dunaliella salina (a green alga), growth under low light (100 mol of photons per m2 per sec) entails damage, degradation, and replacement of D1 every 7 hr. Growth under irradiance stress (2200 micromol of photons per m2 per sec) entails damage to D1 every 20 min. The rate of de novo D1 biosynthesis under conditions of both low light and irradiance stress was found to be fairly constant on a per chloroplast or cell basis. The response of D. salina to the enhanced rate of damage entails an accumulation of photodamaged centers (80% of all PSII) and the formation of thylakoid membranes containing a smaller quantity of photosystem I (PSI) centers (about 10% of that in cells grown under low light). These changes contribute to a shift in the PSII/PSI ratio from 1.4:1 under low-light conditions to 15:1 under irradiance stress. The accumulation of photodamaged PSII under irradiance stress reflects a chloroplast inability to match the rate of D1 degradation or turnover with the rate of damage for individual PSII complexes. The altered thylakoid membrane organization ensures that a small fraction of PSII centers remains functional under irradiance stress and sustains electron flow from H2O to ferredoxin with rates sufficient for chloroplast photosynthesis and cell growth.
Replicative intermediates of human papillomavirus type 11 in laryngeal papillomas: site of replication initiation and direction of replication.
Authors: Auborn K J , Little R D , Platt T H , Vaccariello M A , Schildkraut C L
Published: May 2006

We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180 degrees opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.
1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells.
Authors: Hara K , Yonezawa K , Sakaue H , Ando A , Kotani K , Kitamura T , Kitamura Y , Ueda H , Stephens L , Jackson T R
Published: May 2006

Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits. This interaction takes place via the phosphorylated YMXM motifs of IRS-1 and the Src homology region 2 (SH2) domains of p85. In this study, the stable overexpression in a Chinese hamster ovary (CHO) cell line of a mutant p85 alpha (delta p85) protein, which lacks a binding site for p110, disrupted the complex formation between IRS-1 and the catalytic subunit of PI 3-kinase in intact cells during insulin stimulation. Activation of insulin receptor kinase and the tyrosine phosphorylation of IRS-1 remained unaffected. In this cell line, both insulin-stimulated accumulation of phosphatidylinositol 3,4,5-trisphosphate and the insulin-stimulated glucose uptake due to the translocation of GLUT1 glucose transporters were markedly impaired, whereas neither phorbol 12-myristate 13-acetate-stimulated glucose uptake nor the insulin-stimulated activation of RAS was impaired. These results suggest that PI 3-kinase is required for glucose transport in insulin signaling in CHO cells.
Detection of exocyclic 1,N2-propanodeoxyguanosine adducts as common DNA lesions in rodents and humans.
Authors: Nath R G , Chung F L
Published: May 2006

Exocyclic adducts are unique DNA modifications resulting from binding at two sites of bases that normally are involved in hydrogen-bonding for maintaining the double-helical structure of DNA. These adducts have been shown to be formed in rodents upon exposure to carcinogens. Using a sensitive 32P-postlabeling method combined with high performance liquid chromatography, we obtained evidence that 1,N2-propanodeoxyguanosine adducts of acrolein (AdG) and crotonaldehyde (CdG) are present in the liver DNA of humans and rodents without carcinogen treatment. The identities of these adducts were verified by cochromatography with the synthetic adduct standards. Further proof of identities was obtained by conversion mediated by nuclease P1 of the labeled AdG and CdG 3',5'-bisphosphates to their corresponding 5'-monophosphates. This treatment converted the in vivo adducts into products that again cochromatographed in a characteristic pattern with the synthetic 5'-monophosphates of AdG and CdG. Using this assay, we also demonstrated the in vivo stereoselective formation of one of the AdG isomers. The estimated total levels of modification were 1.0-1.7, 0.2-1.0, and 0.3-2.0 adducts in 10(6) guanine bases in the liver DNA of mice, rats, and humans, respectively. The detection of these adducts in relatively high levels without carcinogen treatment suggests that the endogenous factors such as lipid peroxidation may be important for their formation. This study provides evidence for the presence of acrolein- and crotonaldehyde-derived exocyclic adducts as common lesions in the liver DNA of rodents and humans.
Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis.
Authors: Bate N J , Orr J , Ni W , Meromi A , Nadler-Hassar T , Doerner P W , Dixon R A , Lamb C J , Elkind Y
Published: May 2006

Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanoid synthesis. The role of PAL in pathway regulation was investigated by measurement of product accumulation as a function of enzyme activity in a collection of near-isogenic transgenic tobacco plants exhibiting a range of PAL levels from wild type to 0.2% of wild type. In leaf tissue, PAL level is the dominant factor regulating accumulation of the major product chlorogenic acid and overall flux into the pathway. In stems, PAL at wild-type levels contributes, together with downstream steps, in the regulation of lignin deposition and becomes the dominant, rate-determining step at levels 3- to 4-fold below wild type. The metabolic impact of elevated PAL levels was investigated in transgenic leaf callus that overexpressed PAL. Accumulation of the flavonoid rutin, the major product in wild-type callus, was not increased, but several other products accumulated to similarly high levels. These data indicate that PAL is a key step in the regulation of overall flux into the pathway and, hence, accumulation of major phenylpropanoid products, with the regulatory architecture of the pathway poised so that downstream steps control partitioning into different branch pathways.
The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA.
Authors: Stasiak A , Tsaneva I R , West S C , Benson C J , Yu X , Egelman E H
Published: May 2006

The RuvB protein is induced in Escherichia coli as part of the SOS response to DNA damage. It is required for genetic recombination and the postreplication repair of DNA. In vitro, the RuvB protein promotes the branch migration of Holliday junctions and has a DNA helicase activity in reactions that require ATP hydrolysis. We have used electron microscopy, image analysis, and three-dimensional reconstruction to show that the RuvB protein, in the presence of ATP, forms a dodecamer on double-stranded DNA in which two stacked hexameric rings encircle the DNA and are oriented in opposite directions with D6 symmetry. Although helicases are ubiquitous and essential for many aspects of DNA repair, replication, and transcription, three-dimensional reconstruction of a helicase has not yet been reported, to our knowledge. The structural arrangement that is seen may be common to other helicases, such as the simian virus 40 large tumor antigen.
Use of a macromolecular crowding agent to dissect interactions and define functions in transcriptional activation by a DNA-tracking protein: bacteriophage T4 gene 45 protein and late transcription.
Authors: Sanders G M , Kassavetis G A , Geiduschek E P
Published: May 2006

We have used a molecular crowding reagent to define functions in the transcriptional activation of bacteriophage T4 late genes. This activation normally requires the three T4 DNA polymerase accessory proteins encoded by T4 genes 44, 62, and 45 (the gp44/62 complex and gp45), an enhancer-like cis-acting site, an RNA polymerase-bound coactivator, and an unobstructed path along the DNA joining the promoter to the enhancer. We show that molecular crowding eliminates the requirement for the gp44/62 complex and for the enhancer, retains the requirement for gp45 and its coactivator, and generates activated promoter complexes with nearly unchanged DNase I footprints. These experiments identify gp45 as the direct activator of transcription, and the gp44/62 complex as the assembly factor for gp45. They suggest that the enhancer serves as the normal, but not invariably essential, entry site for the gp45 DNA-tracking protein.
Protein kinases C-beta and C-epsilon link the mast cell high-affinity receptor for IgE to the expression of c-fos and c-jun.
Authors: Razin E , Szallasi Z , Kazanietz M G , Blumberg P M , Rivera J
Published: May 2006

In this report we identify the specific isozymes of protein kinase C (PKC) that are involved in c-fos and c-jun mRNA accumulation in the rat basophilic leukemia cell line RBL-2H3. These cells could be largely depleted of the endogenous PKC isozymes by chronic treatment with phorbol 12-myristate 13-acetate followed by permeabilization of the cells with streptolysin O. The reconstitution of these cells with defined concentrations of either PKC-beta or PKC-epsilon up to 10 nM and 20 nM, respectively, induced c-fos and c-jun in a dose-dependent manner. At high concentrations of PKC-beta and -epsilon the induction of c-fos and c-jun was independent of the aggregation of the high-affinity IgE receptors (Fc epsilon type I receptors). In contrast, at limiting concentrations of these two PKC isozymes, 1 nM, the increase in c-fos and c-jun mRNAs was dependent on the aggregation of the Fc epsilon type I receptors. Unlike PKC-beta and -epsilon, PKC-alpha and PKC-delta failed to reconstitute c-fos and c-jun induction at any dose over the range examined. We conclude that PKC-beta and PKC-epsilon serve as a link between the cell surface receptor and gene expression.
A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity.
Authors: Nishikawa R , Ji X D , Harmon R C , Lazar C S , Gill G N , Cavenee W K , Huang H J
Published: May 2006

The development and neoplastic progression of human astrocytic tumors appears to result through an accumulation of genetic alterations occurring in a relatively defined order. One such alteration is amplification of the epidermal growth factor receptor (EGFR) gene. This episomal amplification occurs in 40-50% of glioblastomas, which also normally express endogenous receptors. Moreover, a significant fraction of amplified genes are rearranged to specifically eliminate a DNA fragment containing exons 2-7 of the gene, resulting in an in-frame deletion of 801 bp of the coding sequence of the extracellular domain. Here we used retroviral transfer of such a mutant receptor (de 2-7 EGFR) into glioblastoma cells expressing normal endogenous receptors to test whether the mutant receptor was able to augment their growth and malignancy. Western blotting analysis showed that these cells expressed endogenous EGFR of 170 kDa as well as the exogenous de 2-7 EGFR of 140-155 kDa. Although holo-EGFRs were phosphorylated on tyrosine residues only after exposure of the cells to ligand, de 2-7 EGFRs were constitutively phosphorylated. In tissue culture neither addition of EGF nor expression of the mutant EGFR affected the rate of cell growth. However, when cells expressing mutant EGFR were implanted into nude mice subcutaneously or intracerebrally, tumorigenic capacity was greatly enhanced. These results suggest that a tumor-specific alteration of the EGFR plays a significant role in tumor progression perhaps by influencing interactions of tumor cells with their microenvironment in ways not easily assayed in vitro.
The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes.
Authors: Rudd M D , Izban M G , Luse D S
Published: May 2006

RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SII increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SII, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionally active.
Leucine-zipper dimerization motif encoded by the AF17 gene fused to ALL-1 (MLL) in acute leukemia.
Authors: Prasad R , Leshkowitz D , Gu Y , Alder H , Nakamura T , Saito H , Huebner K , Berger R , Croce C M , Canaani E
Published: May 2006

Chromosome region 11q23 is involved in reciprocal chromosome translocations associated with human acute leukemias. These aberrations fuse the ALL-1 gene located at 11q23 to a series of partner genes positioned on a variety of human chromosomes. The fused genes encode chimeric proteins. Here we report the cloning and characterization of the ALL-1 partner at 17q21, the AF17 gene. The AF17 gene encodes a protein of 1093 amino acids, containing a leucine-zipper dimerization motif located 3' of the fusion point and a cysteine-rich domain at the N terminus. The latter can be arranged in three zinc fingers and shows homology to a domain within the protein Br140 (peregrin). AF17 contains stretches of amino acids previously associated with domains involved in transcriptional repression or activation. Based on features of AF17 and of the proteins encoded by the other partner genes analyzed and in conjunction with other recent studies, we propose a model in which ALL-1 rearrangements result in loss of function of the gene. In this model, the partner polypeptide plays an accessory role either by repressing activity of the truncated ALL-1 protein or by blocking the function of the normal protein presumably present in the leukemic cells.
Binding of Bruton's tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-mediated interaction.
Authors: Cheng G , Ye Z S , Baltimore D
Published: May 2006

Bruton's tyrosine kinase (Btk) is a recently described B-cell-specific tyrosine kinase. Mutations in this gene lead to human X chromosome-linked agammaglobulinemia and murine X-linked immunodeficiency. Although genetic evidence strongly suggests that Btk plays a crucial role in B-lymphocyte differentiation and activation, its precise mechanism of action remains unknown, primarily because the proteins that it interacts with have not yet been identified. Here, we show that Btk interacts with Src homology 3 domains of Fyn, Lyn, and Hck, protein-tyrosine kinases that get activated upon stimulation of B- and T-cell receptors. These interactions are mediated by two 10-aa motifs in Btk. An analogous site with the same specificity is also present in Itk, the T-cell-specific homologue of Btk. Our data extend the range of interactions mediated by Src homology 3 domains and provide an indication of a link between Btk and established signaling pathways in B lymphocytes.