bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium.
Authors: Abou-Samra A B , Jüppner H , Force T , Freeman M W , Kong X F , Schipani E , Urena P , Richards J , Bonventre J V , Potts J T
Published: May 2006

Parathyroid hormone (PTH), a major regulator of mineral ion metabolism, and PTH-related peptide (PTHrP), which causes hypercalcemia in some cancer patients, stimulate multiple signals (cAMP, inositol phosphates, and calcium) probably by activating common receptors in bone and kidney. Using expression cloning, we have isolated a cDNA clone encoding rat bone PTH/PTHrP receptor from rat osteosarcoma (ROS 17/2.8) cells. The rat bone PTH/PTHrP receptor is 78% identical to the opossum kidney receptor; this identity indicates striking conservation of this receptor across distant mammalian species. Additionally, the rat bone PTH/PTHrP receptor has significant homology to the secretin and calcitonin receptors but not to any other G protein-linked receptor. When expressed in COS cells, a single cDNA clone, expressing either rat bone or opossum kidney PTH/PTHrP receptor, mediates PTH and PTHrP stimulation of both adenylate cyclase and phospholipase C. These properties could explain the diversity of PTH action without the need to postulate other receptor subtypes.
Mutant p53 can induce tumorigenic conversion of human bronchial epithelial cells and reduce their responsiveness to a negative growth factor, transforming growth factor beta 1.
Authors: Gerwin B I , Spillare E , Forrester K , Lehman T A , Kispert J , Welsh J A , Pfeifer A M , Lechner J F , Baker S J , Vogelstein B
Published: May 2006

Loss of normal functions and gain of oncogenic functions when the p53 tumor suppressor gene is mutated are considered critical events in the development of the majority of human cancers. Human bronchial epithelial cells (BEAS-2B) provide an in vitro model system to study growth, differentiation, and neoplastic transformation of progenitor cells of lung carcinoma. When wild-type (WT) or mutant (MT; codon 143Val-Ala) human p53 cDNA was transfected into nontumorigenic BEAS-2B cells, we observed that (i) transfected WT p53 suppresses and MT p53 enhances the colony-forming efficiency of these cells, (ii) MT p53 increases resistance to transforming growth factor beta 1, and (iii) clones of MT p53 transfected BEAS-2B cells are tumorigenic when inoculated into athymic nude mice. These results are consistent with the hypothesis that certain mutations in p53 may function in multistage lung carcinogenesis by reducing the responsiveness of bronchial epithelial cells to negative growth factors.
Epinephrine suppresses rap1B.GAP-activated GTPase activity in human platelets.
Authors: Marti K B , Lapetina E G
Published: May 2006

Lysate from quiescent platelets promotes rapid hydrolysis of [gamma-32P]GTP bound to rap1B. Various platelet agonists, including platelet-activating factor, phorbol 12,13-dibutyrate, alpha-thrombin, epinephrine, ADP, and iloprost, that affect platelet metabolism by different signal transduction pathways were used to stimulate intact platelets and study their effects on rap1B.GAP-activated GTPase activity (GAP, GTPase-activating protein). Only epinephrine was found to dramatically decrease not only the rate but also the amount of hydrolysis of rap1B-bound GTP activated by rap1B.GAP. This effect was dose dependent and occurred rapidly. The suppression of GTPase activity was specific for rap1B.GAP in that ras.GAP- and rap2B.GAP-activated GTPase activity were not affected by epinephrine stimulation. This effect appears to be mediated by the alpha 2-adrenergic receptor, as evidenced by a similar suppression of GTPase activity by stimulating platelets with the synthetic alpha 2-adrenergic receptor agonist UK14304 (bromoxidine). Furthermore, the selective alpha 2-adrenergic receptor antagonist yohimbine blocked the suppression of GTPase activity expressed in epinephrine-stimulated cell lysates. No apparent changes in the patterns of protein expression or tyrosine phosphorylation were observed. Although the migration characteristics upon anion-exchange chromatography of rap1B.GAP and ras.GAP activities were unaffected by epinephrine stimulation, the specific activity of rap1B.GAP was noticeably decreased with 250 and 500 microM epinephrine. These results suggest a possible role for rap1B and rap1B.GAP in epinephrine-stimulated signal transduction.
Expression of the mouse tyrosinase gene during embryonic development: recapitulation of the temporal regulation in transgenic mice.
Authors: Beermann F , Schmid E , Schütz G
Published: May 2006

Pigment in mammals is produced in melanocytes of the skin derived from the neural crest and in the pigmented epithelial cells of the retina derived from the optic cup. Tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) is regarded as the key enzyme in pigment synthesis in both these cell types. In this study, we have investigated the temporal regulation of expression of the tyrosinase gene during early eye development and in the developing hair follicle of the mouse by in situ hybridization and have asked whether transgenes would precisely mimick this pattern. We show that the mouse tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression in melanocytes is detected from day 16.5 onwards. This cell type-specific and temporal expression is largely reproduced in transgenic mice carrying a tyrosinase minigene. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell-type specificity and developmental regulation in melanocytes and in the pigment epithelium.
The human cytokine I-309 is a monocyte chemoattractant.
Authors: Miller M D , Krangel M S
Published: May 2006

The human cytokine I-309 is a small glycoprotein secreted by activated T lymphocytes and structurally related to a number of inflammatory cytokines. To investigate the biological activities of I-309 protein, we produced a stable Chinese hamster ovary cell transfectant, CDI.10, which constitutively secretes I-309 protein into culture supernatant. Affinity chromatography on a heparin-Sepharose matrix followed by reverse-phase HPLC was used to purify to homogeneity a glycoprotein doublet of 15-16 kDa from culture supernatant. Biochemical analysis showed the purified recombinant I-309 glycoprotein to be indistinguishable from the natural I-309 glycoprotein constitutively secreted by the T-cell line IDP2. Purified recombinant I-309 stimulated migration of human monocytes but not neutrophils when tested by in vitro chemotaxis assay. Furthermore, the purified protein transiently increased cytoplasmic free calcium concentration in human peripheral blood monocytes but did not do so in lymphocytes or neutrophils. These results demonstrate that the I-309 gene encodes an inflammatory mediator that specifically stimulates human monocytes.
Oral antilymphocyte activity and induction of apoptosis by 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine.
Authors: Carson D A , Wasson D B , Esparza L M , Carrera C J , Kipps T J , Cottam H B
Published: May 2006

2-Chlorodeoxyadenosine (CdA) is active in chronic lymphocytic leukemia, hairy-cell leukemia, and low-grade lymphomas. In part, this spectrum of activity may be attributable to the selective toxicity of CdA to nondividing lymphocytes and monocytes. However, CdA is unstable at acidic pH and is degraded by bacterial nucleoside phosphorylases. The present experiments demonstrate that the 2'-arabino-fluoro derivative of CdA, designated CAFdA, is also directly toxic to quiescent lymphocytes and macrophages. Unlike CdA, CAFdA was stable at pH 2 and resisted degradation by Escherichia coli nucleoside phosphorylase. Cell killing was preceded by the formation of DNA strand breaks and could be prevented by supplementation of the medium with deoxycytidine. The initial DNA damage initiated the pattern of oligonucleosomal DNA fragmentation characteristic of apoptosis. Mutant lymphoblasts, deficient in deoxycytidine kinase, with elevated cytoplasmic 5'-nucleotidase, or with expanded deoxynucleotide pools secondary to increased ribonucleotide reductase activity, were cross-resistant to both CAFdA and CdA toxicity. One-week oral treatment with CAFdA (1 mg/ml in drinking water) achieved an average plasma concentration of 0.56 microM and eliminated 90% of chronic lymphocytic leukemia cells transplanted into severe combined immunodeficiency (scid) mice. Under the same conditions, CdA was much less active. Collectively, these results suggest that CAFdA could be effective as an oral agent in indolent lymphoproliferative diseases and in autoimmune diseases where lymphocyte and monocyte depletion is desirable.
Protein phosphorylation regulates secretion of Alzheimer beta/A4 amyloid precursor protein.
Authors: Caporaso G L , Gandy S E , Buxbaum J D , Ramabhadran T V , Greengard P
Published: May 2006

Extracellular deposition of the beta/A4 amyloid peptide is a characteristic feature of the brain in patients with Alzheimer disease. beta/A4 amyloid is derived from the amyloid precursor protein (APP), an integral membrane protein that exists as three major isoforms (APP695, APP751, and APP770). Secreted forms of APP found in blood plasma and cerebrospinal fluid arise by proteolytic cleavage of APP within the beta/A4 amyloid domain, precluding the possibility of amyloidogenesis for that population of molecules. In the present study, we have demonstrated that treatment of PC12 cells with phorbol ester produces a severalfold increase in secretion of APP695, APP751, and APP770. This increase is augmented by simultaneous treatment with the protein phosphatase inhibitor okadaic acid. These data indicate that protein phosphorylation regulates intra-beta/A4 amyloid cleavage and APP secretion. These and other results suggest that APP molecules can normally follow either of two processing pathways: regulated secretion or proteolytic degradation unassociated with secretion.
The nonobese diabetic scid mouse: model for spontaneous thymomagenesis associated with immunodeficiency.
Authors: Prochazka M , Gaskins H R , Shultz L D , Leiter E H
Published: May 2006

Homozygosity for the severe combined immunodeficiency (scid) mutation results in a block in T- and B-lymphocyte development. An unusually high incidence of spontaneous thymic lymphoma development was observed after transfer of this mutation from the C.B-17 congenic strain background onto the diabetes-susceptible nonobese diabetic (NOD) background. Thymomagenesis in the NOD-scid/scid mouse was associated with expression of an NOD mouse-unique endogenous ecotropic murine leukemia provirus locus (Emv-30, mapped to proximal region of chromosome 11) not expressed in the standard substrain NOD/Lt thymus. All tumors exhibited insertions of ecotropic proviruses, whereas only a subset also exhibited proviral integrations of mink cell focus-forming retrovirus. Neither class of retrovirus was associated with consistent integration into genes previously associated with activation of oncogenesis. We propose that the unusual features of T-cell ontogeny characteristic of the NOD inbred strain synergize with the scid-imparted block in thymocyte development, leading to activation of the NOD-unique Emv-30 to initiate thymomagenesis.
Human autoantibodies reactive with synthetic autoantigens from T-cell receptor beta chain.
Authors: Marchalonis J J , Kaymaz H , Dedeoglu F , Schluter S F , Yocum D E , Edmundson A B
Published: May 2006

We used mapping with synthetic overlapping peptides in combination with molecular modeling to analyze the IgG antibodies that humans naturally produce against human T-cell receptor beta chains and to localize the recognized peptide autoantigens in the three-dimensional structure of the molecule. Healthy individuals produce low levels of antibodies against T-cell receptor peptides, and these can be increased in autoimmune diseases. We characterized the reactivities in detail because IgG molecules reactive with self peptides occur in preparations of intravenous immunoglobulin and can be isolated by immunoaffinity chromatography. Natural IgG antibodies were directed against three major peptides. One corresponds to the first complementarity-determining region of the variable region. A second corresponds to the third framework of the variable region. The third is located in the constant region and is predicted to be a loop that extends out of the beta-barrel structure. This peptide is one that would give a characteristic structural distinction between the beta-chain constant region and the constant regions of immunoglobulin light chains to which beta chains are homologous. The capacity to bind these peptides is found in small fractions of normal polyclonal IgG, which contains both kappa chains and lambda chains. The activity is antibody-like in being confined to the Fab fragment and in its capacity to discriminate among homologous synthetic peptides corresponding to distinct beta-chain variable-region genes. We propose that a recognition and regulatory process naturally occurs that parallels the immune network for the regulation of the production of antibodies.
Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity.
Authors: Terada L S , Guidot D M , Leff J A , Willingham I R , Hanley M E , Piermattei D , Repine J E
Published: May 2006

Exposure to decreasing oxygen tensions progressively increased xanthine dehydrogenase (XD) and xanthine oxidase (XO) activities over 48 hr in cultured pulmonary artery endothelial cells (EC) without altering XD/XO ratios. Increases in XD and XO activity in EC induced by hypoxia were associated upon reoxygenation with increased (P less than 0.05) extracellular superoxide anion (O2-.) levels that were inhibited by treatment with XO inhibitors (tungsten, allopurinol) or an anion-channel blocker (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid). EC monolayers subjected to hypoxia/reoxygenation also leaked more preloaded 51Cr, were more adherent to neutrophils, and permitted greater albumin transit than control monolayers. Treatment with tungsten, allopurinol, and/or superoxide dismutase decreased (P less than 0.05) 51Cr release, neutrophil adherence, and albumin transit in EC monolayers exposed to hypoxia/reoxygenation. We conclude that prolonged hypoxia increases both XO and XD activity in EC and may predispose the endothelium to oxidative and inflammatory damage.
On the expression and regulation of 5-lipoxygenase in human lymphocytes.
Authors: Jakobsson P J , Steinhilber D , Odlander B , Rådmark O , Claesson H E , Samuelsson B
Published: May 2006

The expression of arachidonate 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) and the 5-lipoxygenase-activating protein (FLAP) genes in human tonsillar B cells and lymphoblastoid B-cell lines was demonstrated at the transcriptional level by reverse transcription-PCR analysis. Also, five lymphoblastoid T-cell lines were investigated and found to express the FLAP gene but not the 5-lipoxygenase gene, suggesting that the transcriptional regulation of these two genes is different. Western blot analysis of the cytosolic proteins from a lymphoblastoid B-cell line with an antiserum raised against purified human leukocyte 5-lipoxygenase revealed an immunoreactive band that comigrated with recombinant human 5-lipoxygenase. Intact B cells produced very low amounts of leukotriene B4 and 5-hydroxyeicosatetraenoic acid upon stimulation with the calcium ionophore A23187 and arachidonic acid, in comparison to the amounts formed by sonicates of these cells. However, preincubation of intact lymphoblastoid B cells with the glutathione-depleting agents azodicarboxylic acid bis(dimethylamide) or 1-chloro-2,4-dinitrobenzene prior to the addition of the calcium ionophore A23187 and arachidonic acid led to similar amounts of leukotriene B4 as were formed by sonicated cells. In contrast, the glutathione synthesis inhibitor buthionine sulfoximine diminished the cellular level of glutathione by greater than 90% but did not influence the production of leukotriene B4 or 5-hydroxyeicosatetraenoic acid in intact cells. These results demonstrate that certain drugs affecting the redox status can stimulate the cryptic 5-lipoxygenase activity in intact lymphoblastoid B cells but that the mechanism of this activation is unclear and appears not to be directly related to intracellular glutathione levels.
Selectively amplified expression of an isoform of the vacuolar H(+)-ATPase 56-kilodalton subunit in renal intercalated cells.
Authors: Nelson R D , Guo X L , Masood K , Brown D , Kalkbrenner M , Gluck S
Published: May 2006

The intercalated cells of the kidney collecting duct are specialized for physiologically regulated proton transport. In these cells, a vacuolar H(+)-ATPase is expressed at enormous levels in a polarized distribution on the plasma membrane, enabling it to serve in transepithelial H+ transport. In contrast, in most eukaryotic cells, vacuolar H(+)-ATPases reside principally in intracellular compartments to effect vacuolar acidification. To investigate the basis for the selective amplification of the proton pump in intercalated cells, we isolated and sequenced cDNA clones for two isoforms of the approximately 56-kDa subunit of the H(+)-ATPase and examined their expression in various tissues. The predicted amino acid sequence of the isoforms was highly conserved in the internal region but diverged in the amino and carboxyl termini. mRNA hybridization to a cDNA probe for one isoform (the "kidney" isoform) was detected only in kidney cortex and medulla, whereas mRNA hybridization to the other isoform of the approximately 56-kDa subunit and to the H(+)-ATPase 31-kDa subunit was found in the kidney and other tissues. Immunocytochemistry of rat kidney with an antibody specific to the kidney isoform revealed intense staining only in the intercalated cells. Staining was absent from proximal tubule and thick ascending limb, where H(+)-ATPase was detected with a monoclonal antibody to the 31-kDa subunit of the H(+)-ATPase. This example of specific amplification of an isoform of one subunit of the vacuolar H(+)-ATPase being limited to a specific cell type suggests that the selective expression of the kidney isoform of the approximately 56-kDa subunit may confer the capacity for amplification and other specialized functions of the vacuolar H(+)-ATPase in the renal intercalated cell.
Calcium uptake in the skin of a freshwater teleost.
Authors: McCormick S D , Hasegawa S , Hirano T
Published: May 2006

The skin, particularly the opercular membrane of some teleosts, contains mitochondrion-rich "chloride" cells and has been widely used as a model to study branchial salt-extrusion mechanisms in seawater fish. Skin isolated from the operculum of the freshwater Nile tilapia (Oreochromis niloticus) can transport Ca2+ against an ionic and electrical gradient. Adaptation of Nile tilapia to a low-Ca2+ environment increased the capacity of the opercular membrane to transport Ca2+. The density of mitochondrion-rich cells increased in parallel with Ca2+ transport capacity. The results demonstrate net Ca2+ uptake by vertebrate skin and strongly implicate mitochondrion-rich cells as the site of Ca2+ uptake in fresh water.
Ribosomal RNA sequences and the diversity of symbiotic dinoflagellates (zooxanthellae).
Authors: Rowan R , Powers D A
Published: May 2006

Zooxanthellae are unicellular algae that occur as endosymbionts in many hundreds of common marine invertebrates. The issue of zooxanthella diversity has been difficult to address. Most zooxanthellae have been placed in the dinoflagellate genus Symbiodinium as one or several species that are not easily distinguished. We compared Symbiodinium and nonsymbiotic dinoflagellates using small ribosomal subunit RNA sequences. Surprisingly, small ribosomal subunit RNA diversity within the genus Symbiodinium is comparable to that observed among different orders of nonsymbiotic dinoflagellates. These data reinforce the conclusion that Symbiodinium-like zooxanthellae represent a collection of distinct species and provide a precedent for a molecular genetic taxonomy of the genus Symbiodinium.
X-ray crystal structures of transforming p21 ras mutants suggest a transition-state stabilization mechanism for GTP hydrolysis.
Authors: Privé G G , Milburn M V , Tong L , de Vos A M , Yamaizumi Z , Nishimura S , Kim S H
Published: May 2006

RAS genes isolated from human tumors often have mutations at positions corresponding to amino acid 12 or 61 of the encoded protein (p21), while retroviral ras-encoded p21 contains substitutions at both positions 12 and 59. These mutant proteins are deficient in their GTP hydrolysis activity, and this loss of activity is linked to their transforming potential. The crystal structures of the mutant proteins are presented here as either GDP-bound or GTP-analogue-bound complexes. Based on these structures, a mechanism for the p21 GTPase reaction is proposed that is consistent with the observed structural and biochemical data. The central feature of this mechanism is a specific stabilization complex formed between the Gln-61 side-chain and the pentavalent gamma-phosphate of the GTP transition state. Amino acids other than glutamine at position 61 cannot stabilize the transition state, and amino acids larger than glycine at position 12 would interfere with the transition-state complex. Thr-59 disrupts the normal position of residue 61, thus preventing its participation in the transition-state complex.
Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5' and the 6th phosphodiester bond 3' to the photodimer.
Authors: Huang J C , Svoboda D L , Reardon J T , Sancar A
Published: May 2006

By using a human cell-free system capable of nucleotide excision repair, a synthetic substrate consisting of a plasmid containing four thymidine dimers at unique locations, and deoxyribonucleoside 5'-[alpha-thio]triphosphates for repair synthesis, we obtained DNA fragments containing repair patches with phosphorothioate linkages. Based on the resistance of these linkages to digestion by exonuclease III and their sensitivity to cleavage by I2, we were able to delineate the borders of the repair patch to single-nucleotide resolution and found an asymmetric patch with sharp boundaries. That the repair patch was produced by filling in a gap generated by an excision nuclease and not by nick-translation was confirmed by the finding that the thymidine dimer was released in a 27- to 29-nucleotide oligomer.
Conserved sequence-tagged sites: a phylogenetic approach to genome mapping.
Authors: Mazzarella R , Montanaro V , Kere J , Reinbold R , Ciccodicola A , D'Urso M , Schlessinger D
Published: May 2006

Cognate sites in genomes that diverged approximately 100 million years ago can be detected by PCR assays based on primer pairs from unique sequences. The great majority of such syntenically equivalent sequence-tagged sites (STSs) from human DNA can be used to assemble and format corresponding maps for other primates, and some based on gene sequences are shown to be useful for mouse and rat as well. Universal genomic mapping strategies may be possible by using sets of STSs common to many mammalian species.
Mouse model for exoerythrocytic stages of Plasmodium falciparum malaria parasite.
Authors: Sacci J B , Schriefer M E , Resau J H , Wirtz R A , Detolla L J , Markham R B , Azad A F
Published: May 2006

Research on the exoerythrocytic (EE) stages of human malaria parasites has been hindered because of the lack of an easily available suitable animal model. We report here an approach to produce mature EE-stage Plasmodium falciparum parasites by using severe combined immunodeficient (scid) mice with transplanted human hepatocytes. Transplantation of human hepatocytes into scid mice (scid hu-hep), their subsequent intravenous infection with P. falciparum sporozoites, and the development of mature liver-stage merozoites was achieved. Immunofluorescent staining of scid hu-hep kidney tissue sections demonstrated the presence of circumsporozoite protein (early during infection), merozoite surface antigen 1, and liver schizont antigen 1. The scid hu-hep model can serve as a source of human malaria liver-stage parasites, decreasing the need for nonhuman primates. Use of this model will facilitate characterization of EE-stage antigens and the assessment of stage-specific chemotherapeutic agents and candidate vaccines.
Oligonucleotide-mediated triple helix formation using an N3-protonated deoxycytidine analog exhibiting pH-independent binding within the physiological range.
Authors: Krawczyk S H , Milligan J F , Wadwani S , Moulds C , Froehler B C , Matteucci M D
Published: May 2006

Triple helix formation with pyrimidine deoxyoligonucleotides for the sequence-specific recognition of DNA duplex targets suffers from a decrease in affinity as the pH of the medium increases to that of physiological fluids. A solution to this problem has been identified and entails the substitution of N6-methyl-8-oxo-2'-deoxyadenosine (M) for the 5-methyl-deoxycytosine base residues. The triple helix forming ability of an oligonucleotide consisting of thymidine and M residues is pH independent in the physiological range. Furthermore, M has been found to be superior to the previously used 5-methyldeoxycytidine and deoxyguanosine in conferring increased affinity for duplex DNA under physiological salt conditions.
Repression of the beta-amyloid gene in a Hox-3.1-producing cell line.
Authors: Violette S M , Shashikant C S , Salbaum J M , Belting H G , Wang J C , Ruddle F H
Published: May 2006

Mammalian homeobox genes are widely expressed in the developing central nervous system and are postulated to control developmental processes by regulating gene expression at the transcriptional level. In vitro studies have identified consensus DNA sequences that contain an ATTA core as sites for interaction with homeodomain proteins. Such elements have been found in the upstream regulatory region of the gene encoding beta-amyloid precursor protein, which is associated with the neurological disorder Alzheimer disease. As the beta-amyloid precursor protein gene is also expressed in the developing central nervous system and appears to play a role in cellular regulatory processes, we have examined the possibility that a homeobox gene product can regulate its transcription. We demonstrate by Northern blot analyses and transfection experiments that the expression of the beta-amyloid precursor protein gene is decreased in cultured cells expressing the mouse homeobox gene Hox-3.1.
Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake, and transcriptional control by iron.
Authors: Dancis A , Roman D G , Anderson G J , Hinnebusch A G , Klausner R D
Published: May 2006

The principal iron uptake system of Saccharomyces cerevisiae utilizes a reductase activity that acts on ferric iron chelates external to the cell. The FRE1 gene product is required for this activity. The deduced amino acid sequence of the FRE1 protein exhibits hydrophobic regions compatible with transmembrane domains and has significant similarity to the sequence of the plasma membrane cytochrome b558 (the X-CGD protein), a critical component of a human phagocyte oxidoreductase, suggesting that FRE1 is a structural component of the yeast ferric reductase. FRE1 mRNA levels are repressed by iron. Fusion of 977 base pairs of FRE1 DNA upstream from the translation start site of an Escherichia coli lacZ reporter gene confers iron-dependent regulation on expression of beta-galactosidase in yeast. An 85-base-pair segment of FRE1 5' noncoding sequence contains a RAP1 binding site and a repeated sequence, TTTTTGCTCAYC; this segment is sufficient to confer iron-repressible transcriptional activity on heterologous downstream promoter elements.
Dissection of the enhancer activity of beta-globin 5' DNase I-hypersensitive site 2 in transgenic mice.
Authors: Liu D , Chang J C , Moi P , Liu W , Kan Y W , Curtin P T
Published: May 2006

The beta-globin locus control region (LCR) consists of four erythroid-specific DNase I-hypersensitive sites, which are necessary for high-level expression of the beta-like globin genes in erythroid tissues. One of these sites, designated 5'HS-2, functions as an erythroid-specific enhancer element in transfection and transgenic mouse experiments. Recent transfection experiments and studies of DNA-protein interactions have localized the 5'HS-2 enhancer to 18 nucleotides that contain a binding site for both the erythroid-specific factor nuclear factor erythroid 2 (NFE-2) and for activator protein 1 (AP-1). To define the sequences necessary for in vivo enhancer activity, several deletion mutants of 5'HS-2 were linked to the human beta-globin gene and their activity was tested in transgenic mice. Three upstream fragments of 5'HS-2 [341, 374, and 412 base pairs (bp)], each of which contained the NFE-2/AP-1 sequences, resulted in beta-globin expression at levels equivalent to or higher than those observed with the entire 732-bp 5'HS-2 fragment. In contrast, a 358-bp downstream portion of 5'HS-2, which lacked the NFE-2/AP-1 sequences, resulted in beta-globin expression at the low levels seen with the beta-globin gene alone. Removal of the NFE-2/AP-1 sequences by a 67-bp internal deletion resulted in similar low levels of beta-globin expression. A 100-bp 5' fragment that contained the NFE-2/AP-1 sequences resulted in beta-globin expression that was higher than the beta-globin gene alone but lower than the entire 5'HS-2 fragment or the three larger upstream fragments. These studies demonstrate that the NFE-2/AP-1 sequences are essential for enhancer activity of 5'HS-2 but that other sequences are required for full activity in vivo.
Allelic variants of the human putative peptide transporter involved in antigen processing.
Authors: Colonna M , Bresnahan M , Bahram S , Strominger J L , Spies T
Published: May 2006

Antigen processing for presentation of peptide epitopes by major histocompatibility complex (MHC) class I molecules involves genes in the MHC class II region. Among these, PSF1 and PSF2 encode subunits of a transporter, which presumably delivers cytosolic peptides across the endoplasmic reticulum membrane to class I molecules. This close functional relationship of the transporter and class I heavy chain genes and their linkage within the MHC raise the question of whether PSF1 and PSF2, like most class I genes, are polymorphic. By single-strand conformation polymorphism analysis and DNA sequencing, a small number of amino acid sequence variants of both PSF1 and PSF2 was identified in a panel of cell lines. This limited polymorphism may contribute to a higher degree of variability at the level of the functional transporter, in which different alleles of the PSF1 and PSF2 subunits may be combined. A possible involvement of the PSF1 and PSF2 genes in susceptibility to MHC-associated diseases was examined in a preliminary assessment in patients with ankylosing spondylitis, insulin-dependent diabetes mellitus, or celiac disease.
A chemical approach to designing Turing patterns in reaction-diffusion systems.
Authors: Lengyel I , Epstein I R
Published: May 2006

A systematic approach is suggested to design chemical systems capable of displaying stationary, symmetry-breaking reaction diffusion patterns (Turing structures). The technique utilizes the fact that reversible complexation of an activator species to form an unreactive, immobile complex reduces the effective diffusion constant of the activator, thereby facilitating the development of Turing patterns. The chlorine dioxide/iodine/malonic acid reaction is examined as an example, and it is suggested that a similar phenomenon may occur in some biological pattern formation processes.
Inverse protein folding problem: designing polymer sequences.
Authors: Yue K , Dill K A
Published: May 2006

We consider the question of how to design proteins. How can we find "good" amino acid sequences (i) that fold to a desired "target" structure as a native conformation of lowest accessible free energy and (ii) that will not simultaneously fold to many other conformations of the same free energy? Current protein designs often focus on helix propensities and turns. We focus here on designing the hydrophobicity. For a model of self-avoiding hydrophobic/polar chains on two-dimensional square lattices, geometric proofs and exhaustive enumerations show the following results. (i) The strategy hydrophobic residues inside/polar residues outside is not optimal. Placement of additional hydrophobic residues on the surface is often necessary. (ii) To avoid unwanted conformations, the designed sequence must have neither too many nor too few hydrophobic residues. (iii) The computational complexity of inverse folding appears to be in a different class than folding: unlike the folding problem, the design problem does not scale exponentially with chain length. Some design strategies, described here for the lattice model, produce good sequences and scale only linearly with chain length.
Intrastriatal transplantation of cross-species fetal striatal cells reduces abnormal movements in a primate model of Huntington disease.
Authors: Hantraye P , Riche D , Maziere M , Isacson O
Published: May 2006

Huntington disease is a neurological movement disorder involving massive neuronal death in the caudate-putamen region of the brain. Neither preventive nor curative therapy exists for this disease. The implantation of cross-species striatal neural precursor cells into the lesioned striatum of nonhuman primates (baboons) reduced the abnormal movements seen in the disease model. These abnormal movements reappeared after immunological rejection of the implanted striatal cells and were not modified by transplantation with nonstriatal cells. These findings encourage further experimentation toward the use of cell sources other than human fetal cells in a potential clinical application to Huntington disease.
Expression and function of the murine B7 antigen, the major costimulatory molecule expressed by peritoneal exudate cells.
Authors: Razi-Wolf Z , Freeman G J , Galvin F , Benacerraf B , Nadler L , Reiser H
Published: May 2006

The murine B7 (mB7) protein is a potent costimulatory molecule for the T-cell receptor (TCR)-mediated activation of murine CD4+ T cells. We have previously shown that stable mB7-transfected Chinese hamster ovary (CHO) cells but not vector-transfected controls synergize with either anti-CD3 monoclonal antibody-induced or concanavalin A-induced T-cell activation, resulting ultimately in lymphokine production and proliferation. We now have generated a hamster anti-mB7 monoclonal antibody. This reagent recognizes a protein with an apparent molecular mass of 50-60 kDa. The mB7 antigen is expressed on activated B cells and on peritoneal exudate cells (PECs). Antibody blocking experiments demonstrate that mB7 is the major costimulatory molecule expressed by PECs for the activation of murine CD4+ T cells. This suggests an important role for mB7 during immune-cell interactions. We have also surveyed a panel of murine cell lines capable of providing costimulatory activity. Our results indicate that mB7 is the major costimulatory molecule on some but not all cell lines and that there may be additional molecules besides mB7 that can costimulate the activation of murine CD4+ T cells.
Population genetics of the fragile-X syndrome: multiallelic model for the FMR1 locus.
Authors: Morton N E , Macpherson J N
Published: May 2006

A model is developed to account for recent molecular observations. It postulates four alleles: normal (N), small rather stable insert (S), larger, unstable insert (Z), and large insert (L). The last-named allele causes the fragile-X phenotype, inactivation of the FMR1 locus by methylation, and mental impairment; the FMR1 locus (for fragile-X mental retardation locus 1) resides in the FRAXA region. When this model is fit to pre-molecular data, the Z allele appears to be no more frequent than L, while the S allele is polymorphic. Predictions of the model are in reasonable agreement with observation and suggest much more powerful tests of molecular data, including the Laird hypothesis that conversion of Z to L does not occur in active X chromosomes.
Pooled-sampling makes high-resolution mapping practical with DNA markers.
Authors: Churchill G A , Giovannoni J J , Tanksley S D
Published: May 2006

A pooled-sample approach to the construction of high-resolution genetic maps is described. The strategy depends on the existence of an easily selectable target locus and the ability to produce large segregating populations. If these requirements are met, the pooled-sample mapping approach allows tightly linked markers (e.g., restriction fragment length polymorphisms) to be mapped relative to the target with a great economy of effort. The recombination fractions among loci can be estimated by the maximum likelihood method and a simple approximate estimator is derived. The order of loci is deduced using a Bayesian statistical framework to yield posterior probabilities for all possible orderings of a marker set. Optimal pooling strategies and the effects of misclassification of selected individuals are discussed and studied by computer simulation. The feasibility of this method is demonstrated by the high-resolution mapping of a region on chromosome 5 of tomato that contains a gene regulating fruit ripening.
Electron microscopic study of supramolecular liquid crystalline polymers formed by molecular-recognition-directed self-assembly from complementary chiral components.
Authors: Gulik-Krzywicki T , Fouquey C , Lehn J
Published: May 2006

Electron microscopic observation provides insight into the nature of the polymeric supramolecular liquid crystalline species (TP2, TU2)n formed by polyassociation of the complementary components TP2 and TU2 derived from D-, L-, or meso-tartaric acid (where T is any form of tartaric acid, D is the D species, and L is the L species) and from pyridine (P) and uracil (U) derivatives. Increasing the concentration of equimolecular solutions of (LP2 + LU2) mixtures leads to the progressive assembly of very long supramolecular-polymolecular entities. The process involves successively nucleation to give small nuclei, growth to filaments, and lateral association to tree-like species, strings, and fibers. The species formed are helical; their helicity is right-handed, induced by the chirality of the components and transferred to the larger entities. The data agree with the formulation of the primary filament as a triple-helical species formed by three helically wound supramolecular strands. (DP2 + DU2) mixtures yield left-handed helical species. The helicity of the materials obtained from complementary components having different chirality is imposed by the U component, being, respectively, right- and left-handed for (DP2 + LU2) and (LP2 + DU2). No helicity is found for the meso compounds. The racemic mixture of all four L and D components yields long superhelices of opposite handedness that coexist in the same sample. This points to the occurrence of spontaneous racemate resolution by chiral selection of the components in the self-assembly of these supramolecular liquid crystalline species. The present results illustrate how extended supramolecular-polymolecular entities build up through molecular-recognition-directed polyassociation of complementary components. They also show that molecular chirality is translated into supramolecular helicity that is expressed at the level of the material on nanometric and micrometric scales.
Molecular structure of a protein-tyrosine/threonine kinase activating p42 mitogen-activated protein (MAP) kinase: MAP kinase kinase.
Authors: Wu J , Harrison J K , Vincent L A , Haystead C , Haystead T A , Michel H , Hunt D F , Lynch K R , Sturgill T W
Published: May 2006

MAP kinases p42mapk and p44mapk participate in a protein kinase cascade(s) important for signaling in many cell types and contexts. Both MAP kinases are activated in vitro by MAP kinase kinase, a protein-tyrosine and threonine kinase. A MAP kinase kinase cDNA was isolated from a rat kidney library by using peptide sequence data we obtained from MAP kinase kinase isolated from rabbit skeletal muscle. The deduced sequence, containing 393 amino acids (predicted mass, 43.5 kDa), is most similar to byr1 (Bypass of ras1), a yeast protein kinase functioning in the mating pathway induced by pheromones in Schizosaccharomyces pombe. An unusually large insert is present in MAP kinase kinase between domains IX and X and may contribute to protein-protein interactions with MAP kinase. Major (2.7 kilobases) and minor (1.7 kilobases) transcripts are widely expressed in rat tissues and appear to be derived from a single gene.
Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase.
Authors: Meurs E F , Galabru J , Barber G N , Katze M G , Hovanessian A G
Published: May 2006

RNA-dependent protein kinase is a M(r) 68,000 protein in human cells (p68 kinase) or a M(r) 65,000 protein in murine cells (p65 kinase). p65/p68 is a serine/threonine kinase induced by interferon treatment and generally activated by double-stranded RNAs. Once activated, the known function of this kinase is inhibition of protein synthesis through phosphorylation of the eukaryotic initiation factor 2. Here we have investigated the potential for tumorigenicity in mice of murine NIH 3T3 clones expressing human p68 kinase, either the wild-type or a mutant inactive kinase with a single amino acid substitution in the invariant lysine-296 in the catalytic domain II. Expression of the mutant p68 kinase was correlated with a malignant transformation phenotype, giving rise to the production of large tumors of at least 1 cm in diameter within 7-12 days in all inoculated mice. In contrast, no tumor growth was observed for several weeks in mice inoculated with NIH 3T3 cell clones expressing either the wild-type recombinant p68 kinase or only the endogenous p65 kinase, the murine analogue of the p68 kinase. These results suggest that functional p65/p68 kinase (recently called PKR), by a still undefined mechanism, may also act as a tumor suppressor. Consequently, one of the pathways by which interferon inhibits tumor growth might be through its capacity to induce the enhanced expression of this kinase.
Bovine pancreatic trypsin inhibitor-trypsin complex as a detection system for recombinant proteins.
Authors: Borjigin J , Nathans J
Published: May 2006

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10(-14) and 1.1 x 10(-13) M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.
Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor.
Authors: Gully D , Canton M , Boigegrain R , Jeanjean F , Molimard J C , Poncelet M , Gueudet C , Heaulme M , Leyris R , Brouard A
Published: May 2006

We describe the characteristics of SR 48692, a selective, nonpeptide antagonist of the neurotensin receptor. In vitro, this compound competitively inhibits 125I-labeled neurotensin binding to the high-affinity binding site present in brain tissue from various species with IC50 values of 0.99 +/- 0.14 nM (guinea pig), 4.0 +/- 0.4 nM (rat mesencephalic cells), 7.6 +/- 0.6 nM (COS-7 cells transfected with the cloned high-affinity rat brain receptor), 13.7 +/- 0.3 nM (newborn mouse brain), 17.8 +/- 0.9 nM (newborn human brain), 8.7 +/- 0.7 nM (adult human brain), and 30.3 +/- 1.5 nM (HT-29 cells). It also displaces 125I-labeled neurotensin from the low-affinity levocabastine-sensitive binding sites but at higher concentrations (34.8 +/- 8.3 nM for adult mouse brain and 82.0 +/- 7.4 nM for adult rat brain). In guinea pig striatal slices, SR 48692 blocks K(+)-evoked release of [3H]dopamine stimulated by neurotensin with a potency (IC50 = 0.46 +/- 0.02 nM) that correlates with its binding affinity. In a cell line derived from a human colon carcinoma (HT-29), SR 48692 competitively antagonizes neurotensin-induced intracellular Ca2+ mobilization with a pA2 (-log Kapp) values of 8.13 +/- 0.03, which is consistent with results obtained in binding studies. Moreover, SR 48692 is devoid of any intrinsic agonist activity. This compound is also active in vivo, since it reverses at low dose (80 micrograms/kg) the turning behavior induced by intrastriatal injection of neurotensin in mice with similar potency whatever the route of administration (i.p. or orally) and with a long duration of action (6 hr). Thus, being a potent and selective neurotensin receptor antagonist, SR 48692 may be considered as a powerful tool for investigating the role of neurotensin in physiological and pathological processes.
Evidence for lack of DNA photoreactivating enzyme in humans.
Authors: Li Y F , Kim S T , Sancar A
Published: May 2006

Photoreactivating enzyme (DNA photolyase; deoxyribocyclobutadipyrimidine pyrimidine-lyase, EC 4.1.99.3) repairs UV damage to DNA by utilizing the energy of near-UV/visible light to split pyrimidine dimers into monomers. The enzyme is widespread in nature but is absent in certain species in a seemingly unpredictable manner. Its presence in humans has been a source of considerable controversy. To help resolve the issue we used a very specific and sensitive assay to compare photoreactivation activity in human, rattlesnake, yeast, and Escherichia coli cells. Photolyase was easily detectable in E. coli, yeast, and rattlesnake cell-free extracts but none was detected in cell-free extracts from HeLa cells or human white blood cells with an assay capable of detecting 10 molecules per cell. We conclude that humans most likely do not have DNA photolyase.
Allosteric mechanism for translational repression in the Escherichia coli alpha operon.
Authors: Spedding G , Draper D E
Published: May 2006

The ribosomal protein S4 is a translational repressor that binds to a complex mRNA pseudoknot structure containing the ribosome binding site for the first gene of the alpha operon. Either 30S subunits or S4 protein bound to the mRNA causes Moloney murine leukemia virus reverse transcriptase to pause near the 3' terminus of the pseudoknot. There is no competition between subunits and S4 for mRNA binding. The kinetics of forming S4-30S-mRNA complexes are biphasic, and the fraction of mRNA molecules reacting more rapidly decreases as the temperature is increased from 30 degrees C to 40 degrees C. The complex cannot be detected with mRNA mutants that cannot be repressed. We have previously shown similar kinetic behavior for the formation of tRNA(fMet) initiation complexes with tRNA(fMet), 30S subunits, and mRNA, except that the fraction reacting rapidly increases when the temperature is increased over the same 30-40 degrees C range. Thus the two sets of experiments show that there are two forms of 30S-mRNA complexes that differ in their abilities to bind S4 and tRNA(fMet). The results support an allosteric model for translational repression in which S4 traps the mRNA in a conformation able to bind 30S subunits but unable to form an initiation complex with tRNA(fMet).