bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of dihydropyridine-sensitive calcium channels in heart cells by fish oil fatty acids.
Authors: Hallaq H , Smith T W , Leaf A
Published: May 2006

The highly unsaturated n-3 fatty acids from fish oils, eicosapentaenoic acid [EPA; C20:5 (n-3)] and docosahexanoic acid [DHA; C22:6 (n-3)], prevent the toxicity of high concentrations of the cardiac glycoside ouabain to isolated neonatal rat cardiac myocytes. Arachidonic acid [C20:4 (n-6)] lacks such protective action. The protective effect of the n-3 fatty acids is associated with their ability to prevent high levels of cytosolic free calcium from occurring in response to the ouabain. This in turn results, at least in part, from a 30% reduction in calcium influx rate induced by the n-3 fatty acids. This protective effect is simulated by nitrendipine, a dihydropyridine inhibitor of the L-type calcium channels in cardiac myocytes. Nitrendipine (0.1 mM) alone, however, inhibits myocyte contractility, as do verapamil (10 microM) and diltiazem (1.0 microM). EPA or DHA (5 microM) blocks the inhibitory effects of nitrendipine but not those of verapamil or diltiazem. Bay K8644, a known dihydropyridine agonist of L-type calcium channels, produces a ouabain-like effect that is also prevented by EPA or DHA. Specific binding of [3H]nitrendipine to intact myocytes is noncompetitively inhibited by EPA or DHA in a manner that reduces the number of high- and low-affinity binding sites (Bmax) and increases their affinities. The fish oil fatty acids prevent calcium overload from ouabain and Bay K8644. They also prevent a calcium-depleted state in the myocytes caused by the L-type calcium channel blocker nitrendipine. The protective effects of the n-3 fatty acids appear to result from their modulatory effects on nitrendipine-sensitive L-type calcium channels.
Early hydrogen-bonding events in the folding reaction of ubiquitin.
Authors: Briggs M S , Roder H
Published: May 2006

The formation of hydrogen-bonded structure in the folding reaction of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a pronounced hydrophobic core, has been investigated by hydrogen-deuterium exchange labeling in conjunction with rapid mixing methods and two-dimensional NMR analysis. The time course of protection from exchange has been measured for 26 back-bone amide protons that form stable hydrogen bonds upon refolding and exchange slowly under native conditions. Amide protons in the beta-sheet and the alpha-helix, as well as protons involved in hydrogen bonds at the helix/sheet interface, become 80% protected in an initial 8-ms folding phase, indicating that the two elements of secondary structure form and associate in a common cooperative folding event. Somewhat slower protection rates for residues 59, 61, and 69 provide evidence for the subsequent stabilization of a surface loop. Most probes also exhibit two minor phases with time constants of about 100 ms and 10 s. Only two of the observed residues, Gln-41 and Arg-42, display significant slow folding phases, with amplitudes of 37% and 22%, respectively, which can be attributed to native-like folding intermediates containing cis peptide bonds for Pro-37 and/or Pro-38. Compared with other proteins studied by pulse labeling, including cytochrome c, ribonuclease, and barnase, the initial formation of hydrogen-bonded structure in ubiquitin occurs at a more rapid rate and slow-folding species are less prominent.
YMXM motifs of IRS-1 define substrate specificity of the insulin receptor kinase.
Authors: Shoelson S E , Chatterjee S , Chaudhuri M , White M F
Published: May 2006

Of 34 tyrosine residues in insulin receptor substrate 1 (IRS-1), 14 are adjacent to acidic residues, suggesting that they might be phosphorylation sites. Synthetic peptides corresponding to sequences surrounding these tyrosines were used as substrates of the insulin receptor kinase. Surprisingly six of these, each within YMXM motifs, were phosphorylated with greatest efficiency (Km, 24-92 microM; kcat/Km, 0.6-2.1 x 10(4) M-1.sec-1). Substituted YMXM peptides revealed a strong preference of the insulin receptor kinase for methionine at Y + 1 and Y + 3 positions. When phosphorylated, related YMXM sequences are recognition motifs for binding to proteins with src-homology (SH2) domains. The combined hydrophobic and flexible nature of methionine side chains adjacent to the targeted tyrosines provides a versatile contact for recognition by diverse proteins involved in signal transduction.
Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene.
Authors: Bu D F , Erlander M G , Hitz B C , Tillakaratne N J , Kaufman D L , Wagner-McPherson C B , Evans G A , Tobin A J
Published: May 2006

We report the isolation and sequencing of cDNAs encoding two human glutamate decarboxylases (GADs; L-glutamate 1-carboxy-lyase, EC 4.1.1.15), GAD65 and GAD67. Human GAD65 cDNA encodes a Mr 65,000 polypeptide, with 585 amino acid residues, whereas human GAD67 encodes a Mr 67,000 polypeptide, with 594 amino acid residues. Both cDNAs direct the synthesis of enzymatically active GADs in bacterial expression systems. Each cDNA hybridizes to a single species of brain mRNA and to a specific set of restriction fragments in human genomic DNA. In situ hybridization of fluorescently labeled GAD probes to human chromosomes localizes the human GAD65 gene to chromosome 10p11.23 and the human GAD67 gene to chromosome 2q31. We conclude that GAD65 and GAD67 each derive from a single separate gene. The cDNAs we describe should allow the bacterial production of test antigens for the diagnosis and prediction of insulin-dependent diabetes mellitus.
A developmentally regulated chlamydial gene with apparent homology to eukaryotic histone H1.
Authors: Perara E , Ganem D , Engel J N
Published: May 2006

We have developed a method for the isolation of genes whose expression is developmentally regulated from the murine strain of Chlamydia trachomatis. Here we describe the identification of two developmental stage-specific genes, one of which is predicted to encode a 26-kDa lysine- and alanine-rich protein that appears to be homologous to several eukaryotic histone H1 proteins. A substantial proportion of this homology relates to its distinctive amino acid composition. No sequence homology was observed between this protein and other bacterial "histone-like" chromosomal proteins, but homology does exist with two other recently described prokaryotic proteins. The protein is expressed late in chlamydial development, during the transition from reticulate bodies to elementary bodies. The basic nature of the protein predicts that it could bind DNA, and Southwestern blotting experiments confirm this finding. These properties are consistent with a role either in the regulation of late gene expression or in the compaction of the chlamydial genome.
Chloroquine inhibits intracellular degradation but not secretion of Alzheimer beta/A4 amyloid precursor protein.
Authors: Caporaso G L , Gandy S E , Buxbaum J D , Greengard P
Published: May 2006

The metabolic fate of the Alzheimer beta/A4 amyloid precursor protein (APP) includes intraamyloid proteolysis that leads to the production of secreted N-terminal and cell-associated C-terminal fragments. The cellular sites at which this processing occurs are not known. We have examined the route of APP processing in metabolically labeled PC12 cells. The lysosomotropic drug chloroquine exerted inhibitory effects on the degradation of mature APP holoprotein. In addition, recovery of a C-terminal fragment resulting from normal intraamyloid cleavage was significantly increased in the presence of chloroquine, suggesting that further degradation of the C-terminal fragment was inhibited. Chloroquine had virtually no effect on APP maturation (N- and O-glycosylation and tyrosine sulfation) or secretion. Treatment with either monensin (which inhibits distal Golgi function) or brefeldin A (which causes resorption of the Golgi into the endoplasmic reticulum and fusion of the trans-Golgi network with the endosomal system) prevented normal APP maturation and abolished APP secretion and recovery of C-terminal fragments, indicating that intact Golgi function is necessary for APP maturation and processing. Our results suggest that a substantial proportion of APP is degraded in an intracellular acidic compartment but that the coupled APP cleavage/secretion event occurs in a chloroquine-insensitive compartment. The observations are consistent with the existence of multiple cellular routes for the trafficking and proteolysis of APP.
Point mutations in the dystrophin gene.
Authors: Roberts R G , Bobrow M , Bentley D R
Published: May 2006

Defining the range of mutations in genes that cause human disease is essential to determine the mechanisms of genetic variation and the function of gene domains and to perform precise carrier and prenatal diagnosis. The mutations in one-third of Duchenne muscular dystrophy patients remain unknown as they do not involve gross rearrangements of the dystrophin gene. The size and complexity of the gene have prohibited the systematic definition of point mutations. We have developed a method for the identification of these mutations by nested amplification, chemical mismatch detection, and sequencing of reverse transcripts of trace amounts of dystrophin mRNA from peripheral blood lymphocytes. Analysis of the entire coding region (11 kilobases) in seven patients has resulted in detection of a sequence change in each case that is clearly sufficient to cause the disease. All mutations should cause premature translational termination, and the resulting phenotypes are thus equivalent to those caused by frameshifting deletions. The results support a particular functional importance for the C-terminal region of dystrophin. Application of this approach to mutation detection will extend direct carrier and prenatal diagnosis to virtually every affected family.
Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures.
Authors: Gundlach H , Müller M J , Kutchan T M , Zenk M H
Published: May 2006

To deter pathogenic microorganisms and herbivores, plants have developed an inducible chemical defense system. It is known that the induced synthesis of low molecular weight compounds can be provoked by exposing cultured cells to fungal cell wall fragments. In this study we show that endogenous jasmonic acid and its methyl ester accumulate rapidly and transiently after treatment of plant cell suspension cultures of Rauvolfia canescens and Eschscholtzia californica with a yeast elicitor. Thirty-six plant species tested in cell suspension culture could be elicited with respect to the accumulation of secondary metabolites by exogenously supplied methyl jasmonate. Addition of methyl jasmonate initiates de novo transcription of genes, such as phenylalanine ammonia lyase, that are known to be involved in the chemical defense mechanisms of plants. These data demonstrate the integral role of jasmonic acid and its derivatives in the intracellular signal cascade that begins with interaction of an elicitor molecule with the plant cell surface and results, ultimately, in the accumulation of secondary compounds.
Rapid detection of inducers of enzymes that protect against carcinogens.
Authors: Prochaska H J , Santamaria A B , Talalay P
Published: May 2006

Dietary composition is a major determinant of cancer risk in humans and experimental animals. Major and minor components of the diet may enhance or suppress the development of malignancy. Many dietary constituents also modify the metabolism of carcinogens by induction of enzymes involved in xenobiotic metabolism, and this is one well-established mechanism for modulating the risk of cancer. We have developed a simple system for rapid detection and measurement of the induction of enzymes that detoxify carcinogens (phase II enzymes), based on the direct assay of the activity of quinone reductase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] in murine hepatoma cells grown in microtiter plate wells. Survey of extracts of a variety of commonly consumed, organically grown vegetables for quinone reductase inducer activity identified crucifers (and particularly those of the genus Brassica) as singularly rich sources. It is therefore of interest that high consumption of these types of vegetables has been correlated with decreased cancer risk in humans. The assay system also measures toxicity, which was unrelated to inducer potency among the vegetable extracts examined. By use of mutant hepatoma cells (defective in regulation of certain cytochrome P-450 enzymes) selective (monofunctional) inducers of protective phase II enzymes can be distinguished from (bifunctional) inducers that also elevate cytochromes P-450 (phase I enzymes) and thereby pose the risk of carcinogen activation. The assay system therefore permits not only rapid detection of inducers of anticarcinogenic enzymes in the human diet but also elucidation of effects of storage and processing on inducer activities.
Sequence of Prochloron didemni atpBE and the inference of chloroplast origins.
Authors: Lockhart P J , Beanland T J , Howe C J , Larkum A W
Published: May 2006

The prochlorophytes, oxygenic photosynthetic prokaryotes containing chlorophylls a and b, have been put forward as descended from the organisms that gave rise to chloroplasts of green plants and algae by endosymbiosis, although this has always been controversial. To assess the phylogenetic position of the prochlorophyte Prochloron didemni, we have cloned and sequenced its atpBE genes. Phylogenetic inference under a range of models gives moderate to strong support for a cyanobacterial grouping rather than a chloroplast one. Possible systematic errors in this and previous analyses of prochlorophyte sequences are discussed.
Origin of Gila seminuda (Teleostei: Cyprinidae) through introgressive hybridization: implications for evolution and conservation.
Authors: DeMarais B D , Dowling T E , Douglas M E , Minckley W L , Marsh P C
Published: May 2006

Morphological and genetic characters from cyprinid fishes of the genus Gila were examined to assess a hypothesized hybrid origin of Gila seminuda from the Virgin River, Arizona-Nevada-Utah. The presumed parents, Gila robusta robusta and Gila elegans, are clearly differentiated from one another based on morphology, allozymes, and mtDNA haplotypes. G. seminuda is morphologically intermediate and polymorphic at allozyme loci diagnostic for the parental species. Restriction endonuclease analysis of mtDNA showed G. seminuda nearly identical to G. elegans. These results support an origin of the bisexual taxon G. seminuda through introgressive hybridization. The Gila population in the Moapa River, Nevada, also appears to be of hybrid origin and is considered a distinctive population of G. seminuda. Inter-specific hybridization is potentially an important mode of evolution among western North American fishes, and valid species of hybrid origin may exist in other groups as well. Consideration of this mode of evolution argues for the need to conserve entire species complexes.
A common antiviral cytotoxic T-lymphocyte epitope for diverse major histocompatibility complex haplotypes: implications for vaccination.
Authors: Oldstone M B , Tishon A , Geckeler R , Lewicki H , Whitton J L
Published: May 2006

Of nine established murine haplotypes, mice of three types (H-2d, H-2u, and H-2q) possess major histocompatibility complex class I glycoproteins able to present an identical viral peptide for recognition and lysis by virus-specific cytotoxic T lymphocytes. Incorporation of this viral epitope into a recombinant vaccinia vaccine and administration of a single dose protects mice with these three haplotypes from an ordinarily lethal challenge of virus. Hence, a common epitope can exist. The sharing of the ability to bind such epitopes among different MHC haplotypes underscores the feasibility of developing an effective cytotoxic T-lymphocyte vaccine for outbred populations like humans.
A protein kinase associated with paired helical filaments in Alzheimer disease.
Authors: Vincent I J , Davies P
Published: May 2006

We have identified a protein kinase in immunoaffinity-purified preparations of paired helical filaments from brain tissue of individuals with Alzheimer disease. The kinase phosphorylates the filament proteins in vitro in a manner independent of second messenger regulation or of modulation by heparin and polyamines. Physiological concentrations of hemin, an oxidized heme porphyrin, inhibit the kinase and abolish Alz-50 immunoreactivity of the proteins. Since paired helical filaments are composed of hyperphosphorylated proteins, association of a protein kinase with the filaments provides a mechanism for abnormal processing of the proteins in disease.
Transactivation and transformation by Myb are negatively regulated by a leucine-zipper structure.
Authors: Kanei-Ishii C , MacMillan E M , Nomura T , Sarai A , Ramsay R G , Aimoto S , Ishii S , Gonda T J
Published: May 2006

The negative regulatory domain of the c-myb protooncogene product (c-Myb) normally represses transcriptional activation by c-Myb. We show here that a leucine-zipper structure is a component of the negative regulatory domain, because its disruption markedly increases both the transactivating and transforming capacities of c-Myb. We also demonstrate that this leucine-zipper structure can interact with cellular proteins. Our results suggest that an inhibitor that suppresses transactivation binds to c-Myb through the leucine zipper and that c-Myb can be oncogenically activated by missense mutation.
Stable growth transformation of human T lymphocytes by herpesvirus saimiri.
Authors: Biesinger B , Müller-Fleckenstein I , Simmer B , Lang G , Wittmann S , Platzer E , Desrosiers R C , Fleckenstein B
Published: May 2006

Herpesvirus saimiri induces T-cell lymphomas in various species of New World monkeys and in rabbits, and it is able to immortalize monkey T lymphocytes in vitro. Sequences responsible for these effects have been localized to a region of the genome that varies significantly among the virus subgroups A, B, and C. We now report that infection of human blood lymphocytes and thymocytes with strains of subgroup C, in contrast to viruses of the other subgroups, yields continuously proliferating T-cell lines with the phenotype of mature CD4- or CD8-positive cells. Infection with strains of Herpes-virus saimiri subgroup C can thus be used to generate human T-cell lines for a variety of immunological and developmental studies.
The gamma 1(34.5) gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of programed cell death in neuronal cells.
Authors: Chou J , Roizman B
Published: May 2006

The gamma 1(34.5) gene of herpes simplex virus 1 was previously shown to play a role in viral virulence since deletion of the gene reduced by a factor of approximately 100,000 the capacity of the virus to replicate in the central nervous system and cause mortality in the mouse. Here we show that in the human neuroblastoma cell line SK-N-SH of neuronal origin gamma 1(34.5) null mutants expressed early proteins, viral DNA, and mRNA of late genes. However, the onset of viral DNA synthesis triggered complete cessation of incorporation of radioactive precursors into proteins. The mutant and wild-type viruses replicated and could not be differentiated in cell lines or cell strains of nonneuronal origin. The results indicate that in the absence of the gamma 1(34.5) gene the SK-N-SH neuroblastoma cells triggered a response similar to the programed cell death of neuronal cells induced by metabolic stress. The gamma 1(34.5) protein precludes this cell response possibly in order to enable the protein synthesis necessary for viral replication.
RHS2, a POU domain-containing gene, and its expression in developing and adult rat.
Authors: Le Moine C , Young W S
Published: May 2006

Gene expression within the central nervous system is regulated by complex interactions of DNA-binding proteins, among which are the POU domain-containing proteins, which are distantly related to homeobox proteins. These POU domain-containing proteins have been implicated in control of transcription and replication within the central nervous system. We used degenerate primers with the PCR to isolate another POU domain-containing cDNA, RHS2, from hypothalamic RNA. Isolation of a putative full-length cDNA was accomplished by using serial dilutions of a hypothalamic cDNA library grown on solid medium. This member of the class III POU family is expressed in rats from embryonic day 11.5 into adulthood, being especially prominent in the brain. We performed double-labeling hybridization histochemistry and determined that RHS2 is coexpressed with a variety of neuropeptides in medium-sized neurons in the caudate putamen and with dynorphin in the paraventricular and supraoptic nuclei of the hypothalamus. Expression of RHS2 in the caudate putamen was increased by elimination of its nigrostriatal dopaminergic innervation.
Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.
Authors: Giavedoni L D , Jones L , Gardner M B , Gibson H L , Ng C T , Barr P J , Yilma T
Published: May 2006

We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41. All fusion proteins retained the antigenic characteristics of both IFN-gamma and HIV as shown by immunoblot analysis. However, the antiviral activity of IFN-gamma could be demonstrated only for the IFN-gamma-gag fusion protein. In contrast, the attenuating activity of IFN-gamma for nude mice was retained by all of the recombinants, albeit at various rates. Unlike the antiviral activity, the attenuating activity of IFN-gamma was not species specific. Implications for the development of attenuated live recombinant vaccines for AIDS are discussed.
Immunoglobulin heavy-chain and CD3 delta-chain gene enhancers are DNase I-hypersensitive in hemopoietic progenitor cells.
Authors: Ford A M , Bennett C A , Healy L E , Navarro E , Spooncer E , Greaves M F
Published: May 2006

Multipotential interleukin 3-dependent non-immortalized murine hemopoietic progenitor cells have DNase I-hypersensitive sites in the immunoglobulin heavy-chain and CD3 delta enhancers and transcribe germ-line T-cell antigen receptor gamma-chain (TCR gamma), but not IgM or TCR beta, genes. Induction of myeloid differentiation in these cells clones down expression and/or transcriptional accessibility of the immunoglobulin heavy-chain and TCR gamma genes. The CD3 delta enhancer region remains DNase I-hypersensitive but closes down in B cells. In embryonic stem cells and pan-mesodermal cells, these genes or enhancer regions are neither expressed nor DNase I-hypersensitive. These data suggest that lineage potential may be programmed, at least in part, by alterations in the accessibility or conformation of regulatory regions of genes and that some promiscuity of gene expression and/or accessibility can precede lineage commitment and maturation in progenitor cells induced to self-renew by interleukin 3.
Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A.
Authors: Shoseyov O , Takagi M , Goldstein M A , Doi R H
Published: May 2006

The cbpA gene for the Clostridium cellulovorans cellulose binding protein (CbpA), which is part of the multisubunit cellulase complex, has been cloned and sequenced. When cbpA was expressed in Escherichia coli, proteins capable of binding to crystalline cellulose and of interacting with anti-CbpA were observed. The cbpA gene consists of 5544 base pairs and encodes a protein containing 1848 amino acids with a molecular mass of 189,036 Da. The open reading frame is preceded by a Gram-positive-type ribosome binding site. A signal peptide sequence of 28 amino acids is present at its N terminus. The encoded protein is highly hydrophobic with extremely high levels of threonine and valine residues. There are two types of putative cellulose binding domains of approximately 100 amino acids that are slightly hydrophilic and eight conserved, highly hydrophobic beta-sheet regions of approximately 140 amino acids. These latter hydrophobic regions may be the CbpA domains that interact with the different enzymatic subunits of the cellulase complex.
Vicinal cysteines in the prosequence play a role in von Willebrand factor multimer assembly.
Authors: Mayadas T N , Wagner D D
Published: May 2006

von Willebrand factor (vWf) is synthesized as a large precursor that dimerizes in the endoplasmic reticulum and forms multimers in the trans- and post-Golgi compartments of megakaryocytes and endothelial cells. The disulfide-bonded multimers are stored in alpha granules of platelets and Weibel-Palade bodies of endothelial cells. The prosequence, composed of two homologous D domains, is required for vWf multimerization and storage. Each D domain contains vicinal cysteines (159Cys-Gly-Leu-162Cys and 521Cys-Gly-Leu-524Cys) that are similar to those at the active site of disulfide isomerases that catalyze thiol protein disulfide interchange. As in disulfide isomerases, a positively charged amino acid (lysine) is also found in close proximity to the vicinal cysteines. Although conserved, the lysine present in thioredoxin was shown not to be essential for its redox activity. We investigated the role of the vicinal cysteines and the lysine residue in the vWf propolypeptide by site-directed mutagenesis and expression of the resulting constructs in mammalian cells. Insertion of an extra glycine between the vicinal cysteines in either D domain inhibited multimerization of dimers, whereas alteration of lysine to glycine in both domains (residues 157 and 519) had no effect. This suggests the importance of the vicinal cysteines but not the lysines in vWf multimerization. Expression of the mutant with an additional glycine in the D1 domain in AtT-20 cells, a mouse pituitary cell line that can store vWf, led to the storage of the resulting dimers. This demonstrates that the mutation did not effect the capacity of the propolypeptide to direct vWf storage while its ability to promote interchain disulfide bonding was eliminated.
A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites.
Authors: Sondek J , Shortle D
Published: May 2006

Results from a number of recent studies suggest that amino acid insertion mutations may provide an important alternative to substitution mutations for modifying protein structures and functional activities. To facilitate the use of single-amino acid insertions, we have developed a general strategy for inducing random, in-phase codon insertions across a defined segment of a cloned gene. In brief, a mixture of blocked and protected trinucleotide phosphoramidites is coupled at substoichiometric levels after every third monomer coupling on a conventional solid-state synthesizer. From the heterogeneous mixture of oligonucleotide sequences thus generated, those oligonucleotides that have acquired a single additional codon are purified by urea/PAGE. By using equimolar amounts of GCT and GGT trinucleotides in the oligonucleotide synthesis plus standard oligonucleotide-directed mutagenesis techniques, we have induced as many as 13 different single alanine and glycine insertion mutations into the gene for staphylococcal nuclease in one experiment. On replacement of the 5'-dimethoxytrityl blocking group on the trinucleotide phosphoramidite with an acid-stable blocking group, such as levulinate or fluoren-9-ylmethoxycarbonyl (Fmoc), this same strategy of substoichiometric couplings at codon boundaries should permit the synthesis of complex pools of oligonucleotides for the introduction, with constant efficiency, of every type of amino acid substitution at each codon across a gene segment.
Expression cloning and characterization of the canine parietal cell gastrin receptor.
Authors: Kopin A S , Lee Y M , McBride E W , Miller L J , Lu M , Lin H Y , Kolakowski L F , Beinborn M
Published: May 2006

Gastrin is an important stimulant of acid secretion by gastric parietal cells and is structurally related to the peptide hormone cholecystokinin (CCK). The pharmacologic properties of the parietal cell gastrin receptor are very similar to the predominant CCK receptor in the brain, CCK-B. Neither the gastrin nor the CCK-B receptor have been cloned thus far, making it difficult to resolve whether these two receptors are distinct. We have isolated a clone encoding the canine gastrin receptor by screening a parietal cell cDNA expression library using a radioligand-binding strategy. Nucleotide sequence analysis revealed an open reading frame encoding a 453-amino acid protein with seven putative hydrophobic transmembrane domains and significant homology with members of the beta-adrenergic family of G protein-coupled receptors. The expressed recombinant receptor shows the same binding specificity for gastrin/CCK agonists and antagonists as the canine parietal cell receptor. Gastrin-stimulated phosphatidylinositol hydrolysis and intracellular Ca2+ mobilization in COS-7 cells expressing the cloned receptor suggest second-messenger signaling through phospholipase C. Affinity labeling of the expressed receptor in COS-7 cells revealed a protein identical in size to the native parietal cell receptor. Gastrin receptor transcripts were identified by high-stringency RNA blot analysis in both parietal cells and cerebral cortex, suggesting that the gastrin and CCK-B receptors are either highly homologous or identical.
Glucocorticoid stabilization of actin filaments: a possible mechanism for inhibition of corticotropin release.
Authors: Castellino F , Heuser J , Marchetti S , Bruno B , Luini A
Published: May 2006

The mechanism by which glucocorticoids induce various cellular responses in different tissues is only partially understood. Here we demonstrate that glucocorticoids stabilize the actin cytoskeleton of several cell types, as revealed by increased resistance of actin filaments to the disrupting effect of cytochalasin and by visible thickening of actin filament bundles. These effects require several hours to develop, require protein synthesis, and are accompanied by increased expression of the actin-binding protein caldesmon. These data may help to explain why glucocorticoids inhibit corticotropin release from pituitary cells, if interpreted in terms of the current idea that an actin filament "barrier" modulates exocytotic secretion in various cell types. In support of this idea, we find that in "model" corticotrophs (AtT-20 cells), glucocorticoids stabilize actin filaments and inhibit corticotropin release with similar potencies. Furthermore, we show here that glucocorticoid inhibition is overcome by exposing AtT-20 cells to concentrations of cytochalasin B or D that disrupt their stabilized actin filaments. On the other hand, our freeze-etch electron microscopy of AtT-20 cells has shown that actin filaments do not, in fact, create a dense submembranous barrier that might prevent corticotropin secretory droplets from discharging; instead, they form open networks near the membrane that appear to hold secretory droplets in their interstices. We propose that the delicate physical crosslinks maintaining this actin-mediated membrane "docking" of secretory droplets may need to disconnect in order to permit corticotropin discharge and that these crosslinks may be stabilized along with the actin filaments in dexamethasone-treated cells.
Cells that present both specific ligand and costimulatory activity are the most efficient inducers of clonal expansion of normal CD4 T cells.
Authors: Liu Y , Janeway C A
Published: May 2006

Clonal expansion of naive CD4 T cells is a necessary step in most adaptive immune responses. Two distinct signals are required for clonal expansion to occur, ligation of T-cell receptors by an antigenic peptide bound to self major histocompatibility complex-encoded class II molecules (signal 1) and a costimulatory signal derived from an antigen-presenting cell (signal 2). To study whether these two signals need to be delivered by a single cell in order to induce clonal expansion of normal CD4 T cells, we have used anti-CD3 bound to Fc receptors as a ligand for the T-cell receptor to deliver signal 1 to all CD4T cells, and we have inactivated signal 2 with a newly generated monoclonal antibody or by using Fc receptor-positive cells that lack the costimulator. Costimulation was delivered by cells whose Fc receptors were blocked with anti-Fc receptor monoclonal antibody. Our results indicate that delivery of ligand and costimulator on one cell is at least 30-fold more efficient than separate delivery. No significant clonal expansion was observed when signals 1 and 2 were delivered by different cells. We have also carried out experiments using fibroblast transfectants that can deliver either or both of these two signals. These studies show that separate delivery of these two signals is at least 80-fold less efficient than their combined delivery by one cell. These findings may explain why tissues can express autoantigens and contain active antigen-presenting cells without inducing autoimmunity.
Differential vulnerability of primate caudate-putamen and striosome-matrix dopamine systems to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
Authors: Moratalla R , Quinn B , DeLanney L E , Irwin I , Langston J W , Graybiel A M
Published: May 2006

The meperidine analogue derivative 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces nigrostriatal fiber damage and severe parkinsonism in humans and animals. MPTP-induced parkinsonism has been proposed as a model of Parkinson disease, but doubts have been raised about whether the patterns of nigrostriatal fiber loss in the two conditions are similar. We report here observations on [3H]mazindol monoamine (principally dopamine) uptake-site binding in the striatum of monkeys (Saimiri sciureus) exposed to low doses of MPTP. We show that this treatment can produce a pattern of nigrostriatal degeneration characteristic of that seen in Parkinson disease, in which there is greater depletion of dopaminergic markers in the putamen than in the caudate nucleus, especially posteriorly. Moreover, within the regions of diminished uptake-site binding in the MPTP-treated monkeys, there is differential preservation of binding in striosomes relative to the surrounding matrix. We suggest that both regional and striosome/matrix patterns of nigrostriatal depletion are key features of MPTP-induced neurodegeneration and that both patterns may provide clues to the mechanisms underlying neurodegeneration in Parkinson disease as well.
Myocardial activation of the human cardiac alpha-actin promoter by helix-loop-helix proteins.
Authors: Sartorelli V , Hong N A , Bishopric N H , Kedes L
Published: May 2006

The cardiac alpha-actin gene is expressed in both heart and skeletal muscle. In skeletal myogenic cells, the 177-base-pair promoter of the human cardiac alpha-actin (HCA) gene requires three transcription factors for activation: Sp1, serum response factor (SRF), and MyoD. However, MyoD is undetectable in heart. To search for a functional equivalent of MyoD, we analyzed the transcriptional regulation of the HCA promoter in primary cultures of rat cardiac myocytes. The same DNA sequence elements recognized by SRF, Sp1, and MyoD and required for HCA transcription in skeletal muscle cells were also found to be necessary for expression in cardiomyocytes. Overexpression of Id, a negative regulator of basic helix-loop-helix proteins, selectively attenuated expression of the HCA promoter. Cardiomyocyte nuclei contain a protein complex that specifically interacts with the same required sequence (E box) in the HCA promoter that is bound by MyoD in skeletal myogenic cells. Furthermore, these complexes contain a peptide that is a member of the E2A family of basic helix-loop-helix proteins. Cardiomyocyte nuclei appear to be enriched for a protein that can bind to the E-box site as dimers with the E12 protein. These results suggest that a member of the basic helix-loop-helix family, together with SRF and Sp1, activates the HCA promoter in heart. Alternative strategies for myocardial transcription of HCA are discussed.
A single gene encodes two isoforms of the p70 S6 kinase: activation upon mitogenic stimulation.
Authors: Reinhard C , Thomas G , Kozma S C
Published: May 2006

Previously, two cDNA clones were isolated from a rat liver or hepatoma cDNA library for the mitogenic-activated p70 S6 kinase (p70s6k). Except for a single amino acid change and a 23-amino acid N-terminal extension in the latter clone, the open reading frames of the two clones are identical. A probe common to both clones also revealed four distinct transcripts. Here, by using specific probes, it was possible to show which transcript corresponds to which clone and that both clones are derived from the same gene. Furthermore, analysis of in vitro translation products using specific antibodies demonstrates that both clones encode the p70s6k but that the clone harboring the 23-amino acid extension also encodes an additional isoform of the kinase, referred to as p85s6k. It could be shown by using the specific antibody to the p85s6k that this isoform of the kinase is present in rat liver and is activated after mitogenic stimulation of quiescent Swiss 3T3 cells.
Synthesis and secretion of wild-type and mutant human plasma cholesteryl ester transfer protein in baculovirus-transfected insect cells: the carboxyl-terminal region is required for both lipoprotein binding and catalysis of transfer.
Authors: Au-Young J , Fielding C J
Published: May 2006

Functional plasma cholesteryl ester transfer protein (CETP; 476 amino acids) has been expressed in baculovirus-transfected Sf9 insect cells by using a full-length cDNA derived from a human placental library. The product bound to each major plasma lipoprotein class, and it catalyzed the transfer of both cholesteryl esters and triglyceride. CETP species with overlapping deletions were generated in the carboxyl-terminal region. These mutants were defective in cholesteryl ester and triglyceride transfer. Structural and functional analysis suggests that normal lipoprotein binding and effective catalysis may require the carboxyl-terminal sequence -Phe-Leu-Leu-Leu- (residues 454-457), possibly with the involvement of other sequences in the carboxyl-terminal region. A similar sequence is contained in several other proteins whose functions involve binding nonpolar lipids, including lecithin: cholesterol acyltransferase, lipopolysaccharide-binding protein, bactericidal permeability-increasing protein, cholesterol 7 alpha-hydroxylase, cholesterol esterase, and hormone-sensitive lipase. These data suggest that a conserved neutral lipid-binding sequence may be one important factor in the activity of CETP and possibly in several other proteins of plasma and cellular lipid metabolism.
Methylation of B-hordein genes in barley endosperm is inversely correlated with gene activity and affected by the regulatory gene Lys3.
Authors: Sørensen M B
Published: May 2006

The methylation status of B-hordein genes in the developing barley endosperm was analyzed by digestion with methylation-sensitive restriction enzymes. Southern blotting revealed specific demethylation of Hpa II sites in DNA from wild-type endosperm, whereas leaf DNA and lys3a mutant endosperm DNA were highly methylated at these sites. Similar methylation patterns were observed at an Ava I site situated at position -260 in the B-hordein promoter. This differential methylation was confirmed by genomic sequencing with ligation-mediated PCR. The analyzed sequence covers most of the B-hordein promoter and includes 10 CpGs from the promoter and 4 CpGs from the adjacent coding region. These sites were all hypomethylated in wild-type endosperm, whereas--except for three partially methylated sites--full methylation was seen in leaf DNA. The four sites in the coding region were partially methylated in lys3a endosperm DNA, but the promoter sites remained highly methylated. The possible role of methylation in the regulatory function of the Lys3 gene product is discussed.
In vivo imaging of calcium accumulation in fly interneurons as elicited by visual motion stimulation.
Authors: Borst A , Egelhaaf M
Published: May 2006

The computation of motion plays a central role in visual orientation. The fly has been successfully used as a model system for analyzing the mechanisms underlying motion detection. Thereby, much attention has been paid to a neuronal circuit of individually identifiable neurons in the third visual ganglion that extracts different types of retinal motion patterns and converts these patterns into specific components of visual orientation behavior. The extended dendritic trees of these large cells are the sites of convergence of numerous spatially distributed local motion-sensitive elements. As is revealed by in vivo microfluorometric imaging, these cells accumulate calcium during activation by visual motion stimulation. The spatiotemporal pattern of calcium distribution shows the following characteristics: (i) calcium accumulation is first spatially restricted to those dendritic branches that are depolarized by the retinotopic input, (ii) during ongoing motion stimulation calcium may also accumulate throughout the cell and, in particular, in regions that do not receive direct synaptic input. These experiments successfully monitor the intracellular distribution of activity-dependent ions in visual interneurons of living animals stimulated by their natural synaptic input.
High rate of diversification and reversal among subclones of neoplastically transformed NIH 3T3 clones.
Authors: Rubin A L , Sneade-Koenig A , Rubin H
Published: May 2006

NIH 3T3 cells undergo neoplastic transformation when exposed to conditions of moderate physiological growth constraint. One of several characteristics of this transformation that indicates its adaptational nature is its gradual reversibility under conditions of unconstrained growth. We explored the origins of reversibility by isolating cells from each of three highly transformed foci and comparing their focus-forming capacity with that of derivative clones and subclones. A high proportion of the parental cells made dense foci. Six of the nine clones obtained from the three foci produced foci, though the percentage varied widely. The other three clones produced no foci at all. The transformed clones were subcloned and analyzed to evaluate the possibility that the negative clones were genuine revertants, rather than being derived from a small minority of nontransformed cells surrounding or underlying the original foci. In each case the subclones varied widely in the percentage of focus-forming cells and the average was much lower than the parental clone from which they were derived. Indeed, 15 of the 53 subclonal populations produced no foci. The high degree of heterogeneity, including complete reversal of focus-forming capacity, provides additional support for the hypothesis that "spontaneous" transformation is driven by an adaptive response to moderate growth constraint rather than by one or more effectively irreversible mutations.
The central distribution of a corticotropin-releasing factor (CRF)-binding protein predicts multiple sites and modes of interaction with CRF.
Authors: Potter E , Behan D P , Linton E A , Lowry P J , Sawchenko P E , Vale W W
Published: May 2006

In recent studies to clone and characterize genes coding for the corticotropin-releasing factor-binding protein (CRF-BP), analysis of the tissue distribution of the CRF-BP gene indicated a high level of expression in the rat brain. We have now characterized by immunohistochemical and hybridization histochemical means the cellular localization of CRF-BP protein and mRNA expression, respectively. Results from both approaches converged to indicate that CRF-BP is expressed predominantly in the cerebral cortex, including all major archi-, paleo-, and neocortical fields. Other prominent sites of mRNA and protein expression include subcortical limbic system structures (amygdala, bed nucleus of the stria terminalis), sensory relays associated with the auditory, olfactory, vestibular, and trigeminal systems, severe raphe nuclei, and a number of cell groups in the brainstem reticular core. Expression in the hypothalamus appears largely limited to the ventral premammillary and dorsomedial nuclei; only isolated CRF-BP-stained cells are apparent in neurosecretory cell groups. Dual immunostaining for CRF and CRF-BP revealed a partial colocalization in some of these regions. In addition, prominent CRF-BP-stained terminal fields have been identified in association with CRF-expressing cell groups in circumscribed hypothalamic and limbic structures. In the anterior pituitary, CRF-BP mRNA and immunoreactivity were colocalized with corticotropin-immunoreactivity in a majority of corticotropes. Thus, CRF-BP could serve to modify the actions of CRF by intra- and intercellular mechanisms, in CRF-related pathways in the central nervous system and pituitary.
The gene encoding the glutamate receptor subunit GluR5 is located on human chromosome 21q21.1-22.1 in the vicinity of the gene for familial amyotrophic lateral sclerosis.
Authors: Eubanks J H , Puranam R S , Kleckner N W , Bettler B , Heinemann S F , McNamara J O
Published: May 2006

Genomic clones of the human non-N-methyl-D-aspartate (non-NMDA) glutamate receptor subunit GluR5 were isolated by high-stringency screening of a cosmid library using the rat cDNA as a probe. The chromosomal localization of the human GluR5 gene has been established. Southern hybridization of DNA isolated from mapping panels of Chinese hamster-human hybrid cell lines and high-resolution in situ suppression hybridization localize the GluR5 gene to chromosome 21q21.1-22.1. This coincides with the localization of a mutant gene causing familial amyotrophic lateral sclerosis (ALS), as Siddique et al. established by linkage analyses [Siddique, T., Figlewicz, D. A., Pericak-Vance, M. A., Haines, J. L., Rouleau, G., Jeffers, A. J., Sapp, P., Hung, W. Y., Bebout, J., McKenna-Yasek, D., Deng, G., Horvitz, H. R., Gusella, J. F., Brown, R. H. & Roses, A. D. (1991) N. Engl. J. Med. 324, 1381-1384]. Convergent evidence from other investigators suggests that chronic pathologic activation of motor neurons via non-NMDA glutamate receptors might induce excitotoxic injury of motor neurons, culminating in ALS. Together with the demonstration that GluR5 transcripts are expressed in the ventral horn of the spinal cord, the region in which susceptible motor neurons reside, the chromosomal localization suggests that a mutated GluR5 gene may be responsible for the familial form of ALS.
Endothelial cells produce a substance that inhibits contact activation of coagulation by blocking the activation of Hageman factor.
Authors: Kleniewski J , Donaldson V H
Published: May 2006

Human umbilical vein endothelial cells (HUVECs) produce a property that impairs the generation of coagulant and amidolytic activity initiated when normal human plasma is exposed to glass. This inhibitory property blocks the adsorption of Hageman factor (factor XII) to glass, thereby preventing the activation of Hageman factor, but does not impair the coagulant or amidolytic activity of already activated Hageman factor (factor XIIa). This property in HUVEC lysates could be neutralized by a purified preparation of Hageman factor but not by purified prekallikrein or high molecular mass kininogen. A partially purified inhibitory fraction from cell lysates exhibited a single homogeneous band in SDS/PAGE of approximately 22.5 kDa. Inhibitory activity was also found in concentrates of conditioned media from HUVECs, which also impaired the binding of Hageman factor to a surface; it may not be identical with that found in cell lysates.