bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of DNA binding and retinoic acid binding properties of retinoic acid receptor.
Authors: Yang N , Schüle R , Mangelsdorf D J , Evans R M
Published: May 2006

High-level expression of the full-length human retinoic acid receptor (RAR) alpha and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RAR alpha protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a Kd of 2.1 x 10(-10) M.
Cell cycle-specific effects of lovastatin.
Authors: Jakóbisiak M , Bruno S , Skierski J S , Darzynkiewicz Z
Published: May 2006

Lovastatin (LOV), the drug recently introduced to treat hypercholesteremia, inhibits the synthesis of mevalonic acid. The effects of LOV on the cell cycle progression of the human bladder carcinoma T24 cell line expressing activated p21ras were investigated. At a concentration of 2-10 microM, LOV arrested cells in G1 and also prolonged--or arrested a minor fraction of cells in--the G2 phase of the cell cycle; at a concentration of 50 microM, LOV was cytotoxic. The cytostatic effects were reversed by addition of exogenous mevalonate. Cells arrested in the cycle by LOV were viable for up to 72 hr and did not show any changes in RNA or protein content or chromatin condensation, which would be typical of either unbalanced growth or deep quiescence. The expression of the proliferation-associated nuclear proteins Ki-67 and p105 in these cells was reduced by up to 72% and 74%, respectively, compared with exponentially growing control cells. After removal of LOV, the cells resumed progression through the cycle; they entered S phase asynchronously after a lag of approximately 6 hr. Because mevalonate is essential for the posttranslational modification (isoprenylation) of p21ras, which in turn allows this protein to become attached to the cell membrane, the data suggest that the LOV-induced G1 arrest may be a consequence of the loss of the signal transduction capacity of p21ras. Indeed, while exposure of cells to LOV had no effect on the cellular content of p21ras (detected immunocytochemically), it altered the intracellular location of this protein, causing its dissociation from the cell membrane and translocation toward the cytoplasm and nucleus. However, it is also possible that inhibition of isoprenylation of proteins other than p21ras (e.g., nuclear lamins) by LOV may be responsible for the observed suppression of growth of T24 cells.
Improved serodiagnosis of hepatitis C virus infection with synthetic peptide antigen from capsid protein.
Authors: Hosein B , Fang C T , Popovsky M A , Ye J , Zhang M , Wang C Y
Published: May 2006

Cloning and expression of hepatitis C virus have allowed the development of immunoassays to detect hepatitis C virus infection. However, currently available recombinant fusion protein C100-3 assays, based on a nonstructural protein of the virus, are limited in sensitivity, particularly for detecting acute infection. In this report seroconversion panels showed that an assay based on synthetic peptides, derived from immunodominant regions of both capsid and nonstructural proteins, accelerated hepatitis C virus antibody detection by 4-10 weeks. In screening, this enzyme immunoassay increased detection from 47% to 64% in plasmapheresis donors with elevated alanine aminotransferase levels (greater than 100 international units per liter), from 15% to 24% in anti-hepatitis B core antigen-positive blood donors, and from 28% to 42% in renal dialysis patients when compared with nonstructural peptide-based assays. The screening assay was repeatedly reactive for 27 of 2902 volunteer blood donor samples (0.93%); four sera reacted only with the capsid antigen. The peptide test distinguished true from false positive results in agreement with recombinant immunoblot assay in 96% of blood donor samples repeatably reactive on a recombinant hepatitis C virus enzyme immunoassay.
Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1.
Authors: Smiley S T , Reers M , Mottola-Hartshorn C , Lin M , Chen A , Smith T W , Steele G D , Chen L B
Published: May 2006

By using a potential-dependent J-aggregate-forming delocalized lipophilic cation, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide (JC-1), we find that membrane potentials across mitochondria in a living cell can be heterogeneous. Remarkably, even within a long contiguous mitochondrion, regional heterogeneity in membrane potentials appears to be possible.
The ATF/CREB transcription factor-binding site in the polymerase beta promoter mediates the positive effect of N-methyl-N'-nitro-N-nitrosoguanidine on transcription.
Authors: Kedar P S , Widen S G , Englander E W , Fornace A J , Wilson S H
Published: May 2006

DNA polymerase beta (pol beta) is a constitutively expressed DNA repair enzyme in vertebrate cells. Yet, it had been shown previously that the pol beta mRNA level increases in Chinese hamster ovary (CHO) cells within 4 h after treatment with several monofunctional DNA damaging agents, notably, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Herein we report that a transfected pol beta promoter fusion gene is activated by MNNG treatment of CHO cells; mRNA from the transfected gene is approximately 10-fold higher in treated cells than in untreated cells 16 h after treatment. This activation is mediated through the decanucleotide palindromic element GTGACGTCAC at positions -49 to -40 in the "TATA-less" core promoter. This element, which is similar to the ATF/CREB transcription factor-binding site in a number of mammalian genes, forms the center of a strong protein-binding site for CHO cell nuclear extract proteins. Mutated pol beta promoter fusion genes lacking the element fail to bind protein at this site and fail to respond to MNNG treatment of cells.
Mutations in conserved yeast DNA primase domains impair DNA replication in vivo.
Authors: Francesconi S , Longhese M P , Piseri A , Santocanale C , Lucchini G , Plevani P
Published: May 2006

To assess the role of eukaryotic DNA primase in vivo, we have produced conditional and lethal point mutations by random in vitro mutagenesis of the PR11 and PR12 genes, which encode the small and large subunits of yeast DNA primase. We replaced the wild-type copies of PRI1 and PRI2 with two pri1 and two pri2 conditional alleles. When shifted to the restrictive temperature, these strains showed altered DNA synthesis and reduced ability to synthesize high molecular weight DNA products, thus providing in vivo evidence for the essential role of DNA primase in eukaryotic DNA replication. Furthermore, mapping of the mutations at the nucleotide level has shown that the two pri1 and two pri2 conditional alleles and one pri2 lethal allele have suffered single base-pair substitutions causing a change in amino acid residues conserved in the corresponding mouse polypeptide.
Bursting electrical activity in pancreatic beta cells caused by Ca(2+)- and voltage-inactivated Ca2+ channels.
Authors: Keizer J , Smolen P
Published: May 2006

We investigate the hypothesis that two classes of Ca2+ currents, one quickly inactivated by Ca2+ and one slowly inactivated by voltage, contribute to bursting electrical activity in pancreatic islets. A mathematical model of these currents is fit to the experimental whole-cell current-voltage and inactivation profiles, thereby fixing the Ca2+ conductance and all activation and inactivation parameters. Incorporating these currents into a model that includes delayed rectifier K+ channels and ATP-sensitive K+ channels, we show that only abnormal bursting is obtained. Modification of activation parameters to increase Ca2+ channel open times, as suggested by experiment, yields a more robust bursting similar to that observed in intact islets. This reinforces the suggestion that in addition to ATP-sensitive K+ channels, Ca2+ channels may serve as glucose sensors in the beta cell.
Chlamydia trachomatis developmentally regulated protein is homologous to eukaryotic histone H1.
Authors: Hackstadt T , Baehr W , Ying Y
Published: May 2006

Chlamydiae are prokaryotic obligate intracellular parasites that undergo a biphasic life cycle involving an infectious, extracellular form known as elementary bodies and an intracellular, replicating form termed reticulate bodies. We have purified from Chlamydia trachomatis a very basic elementary body-specific protein with an apparent molecular mass of 18 kDa, determined its N-terminal amino acid sequence, and cloned the encoding gene. Sequence analysis of the cloned gene revealed some remarkable properties for its expressed product, including a high lysine content (29%), a correspondingly high pI, and significant homology to the H1 class of eukaryotic histones. Furthermore, a monoclonal antibody to this chlamydial histone analog, termed Hc1, displayed immunoblot and antinuclear specificity suggestive of cross-reactivity to H1 histones. The gene was expressed only during the late stages of the chlamydial life cycle concomitant with the reorganization of chlamydial reticulate bodies into elementary bodies, suggesting that the Hc1 protein plays a role in the condensation of chlamydial chromatin during intracellular differentiation.
IgM antigen receptor complex contains phosphoprotein products of B29 and mb-1 genes.
Authors: Campbell K S , Hager E J , Friedrich R J , Cambier J C
Published: May 2006

Membrane immunoglobulin M (mIgM) and mIgD are major B-lymphocyte antigen receptors, which function by internalizing antigens for processing and presentation to T cells and by transducing essential signals for proliferation and differentiation. Although ligation of mIgM or mIgD results in rapid activation of a phospholipase C and a tyrosine kinase(s), these receptors have cytoplasmic tails of only three amino acid residues (Lys-Val-Lys), which seem ill suited for direct physical coupling with cytoplasmic signal transduction structures. In this report, we identify the alpha, beta, and gamma components of the mIgM-associated phosphoprotein complex, which may play a role in signal transduction. Proteolytic peptide mapping demonstrated that the IgM-alpha chain differs from Ig-beta and Ig-gamma. The chains were purified, and amino-terminal sequencing revealed identity with two previously cloned B-cell-specific genes. One component, IgM-alpha, is a product of the mb-1 gene, and the two additional components, Ig-beta and Ig-gamma, are products of the B29 gene. Immunoblotting analysis using rabbit antibodies prepared against predicted peptide sequences of each gene product confirmed the identification of these mIgM-associated proteins. The deduced sequence indicates that these receptor subunits lack inherent protein kinase domains but include common tyrosine-containing sequence motifs, which are likely sites of induced tyrosine phosphorylation.
Antibodies that neutralize human beta interferon biologic activity recognize a linear epitope: analysis by synthetic peptide mapping.
Authors: Redlich P N , Hoeprich P D , Colby C B , Grossberg S E
Published: May 2006

The location of biologically relevant epitopes on recombinant human beta interferon in which Ser-17 replaces Cys-17 (rh[Ser17]IFN-beta) was evaluated by testing the immunoreactivity of antibodies against 159 sequential, overlapping octamer peptides. Three monoclonal antibodies (mAbs) that neutralize rh[Ser17]IFN-beta biologic activity, designated A1, A5, and A7, bound to peptides spanning only residues 39-48, whereas nonneutralizing mAb bound less specifically at multiple sites near the amino terminus. The immunoreactivity of peptides spanning residues 40-47 that contained a series of single amino acid substitutions suggested that residues 41-43 (Pro-Glu-Glu) and 46 (Gln) are important for the binding of neutralizing mAbs. The reactivity of mAbs to larger synthetic peptides containing rh[Ser17]IFN-beta sequences from residue 32 through residue 56 was evaluated. All mAbs except A7 reacted with synthetic peptides representing rh[Ser17]IFN-beta residues 32-47, 40-56, and 32-56, but only mAbs A1 and A5 bound to the core peptide composed of residues 40-47. Peptide 32-56 effectively blocked the binding of mAbs A1 and A5 to rh[Ser17]IFN-beta and markedly inhibited their neutralizing activity. Biologic activity of the peptides was undetectable. Rabbit antisera raised against peptides 32-47 and 40-56 recognized rh[Ser17]IFN-beta but did not neutralize its antiviral activity. Thus, structure-function analysis by peptide mapping has permitted the identification of a linear epitope recognized by neutralizing antibody on a biologically active cytokine. We conclude that the region spanning residues 32-56 is of major importance in the expression of the biologic activity of human IFN-beta.
A heat-labile protein of Chlamydia trachomatis binds to HeLa cells and inhibits the adherence of chlamydiae.
Authors: Joseph T D , Bose S K
Published: May 2006

From highly purified elementary bodies (EBs) of Chlamydia trachomatis, we have identified a protein of 38 kDa that selectively binds to monolayer cultures of HeLa cells. This protein, which we have named the chlamydial cytadhesin (CCA), is present on the surface of the EBs of three C. trachomatis serovars (B, E, and L1) that were examined. Localization of the CCA at the surface was confirmed by its ability to be labeled when viable EBs were iodinated and by its absence in preparations from trypsin-treated EBs. Viable EBs, but not heated or trypsin-treated EBs, inhibited the binding of the CCA to HeLa cells, indicating competition for a common receptor on the host cell membrane. A dose-dependent inhibition of adherence of radioactive EBs to HeLa cells was effected by extracts containing the CCA. This inhibition occurred even with extracts prepared from the EB of heterologous serovars. However, no inhibition could be demonstrated with extracts prepared from heat-treated EBs. Heat treatment of the extract resulted in the loss of ability of the CCA to bind to the host cells. HeLa cells preincubated with CCA-containing chlamydial extract showed reduced ability to bind labeled EBs and to develop cytoplasmic inclusions after infection. This protective activity was lost after exposure of the extract to heat. These findings indicate that the CCA is a thermolabile surface-exposed chlamydial adhesin; it may be useful in the development of vaccines for diseases caused by the pathogenic bacterium.
Synthetic copolymer 1 inhibits human T-cell lines specific for myelin basic protein.
Authors: Teitelbaum D , Milo R , Arnon R , Sela M
Published: May 2006

Copolymer 1 (Cop 1) is a synthetic basic random copolymer of amino acids that has been shown to be effective in suppression of experimental allergic encephalomyelitis and has been proposed as a candidate drug for multiple sclerosis. Cop 1 is immunologically cross reactive with myelin basic protein (BP) and was shown to inhibit murine BP-specific T-cell lines of various H-2 restrictions. In the present study these findings were extended to include human T-cell lines. Cop 1 competitively inhibited the proliferative responses and interleukin 2 secretion of six BP-specific T-cell lines and 13 clones with several DR restrictions and epitope specificities. Conversely, BP inhibited--albeit to a lesser extent--the response of all the Cop 1-specific T-cell lines and clones, irrespective of their DR restrictions. Another random copolymer of tyrosine, glutamic acid, and alanine, denoted TGA, had no effect on these lines. Neither Cop 1 nor BP inhibited the response of lines and clones specific for purified protein derivative. Cop 1 and BP exerted their cross-inhibitory effects only in the presence of antigen-presenting cells. These results suggest that Cop 1 can compete with BP for the binding to human major histocompatibility complex molecules. In view of recent studies implicating BP reactivity in multiple sclerosis, these findings suggest a possible mechanism for the beneficial effect of Cop 1 in this disease.
Functions of signal and signal-anchor sequences are determined by the balance between the hydrophobic segment and the N-terminal charge.
Authors: Sakaguchi M , Tomiyoshi R , Kuroiwa T , Mihara K , Omura T
Published: May 2006

The signal sequence of secretory proteins and the signal-anchor sequence of type II membrane proteins initiate the translocation of the following polypeptide segments, whereas the signal-anchor sequence of cytochrome P-450-type membrane proteins mediates the membrane insertion of the polypeptide via a signal-recognition particle-dependent mechanism but does not lead to the translocation of the following C-terminal sequences. To establish the structural requirements for the function of signal and signal-anchor sequences, we constructed chimeric proteins containing artificial topogenic sequences in which the N-terminal net charge and the length of the hydrophobic segment were systematically altered. Utilizing an in vitro translation-translocation system, we found that hydrophobic segments consisting of 7-10 leucine residues functioned as signal sequences whereas segments with 12-15 leucine residues showed different topogenic functions, behaving as signal sequences or P-450-type signal-anchor sequences, depending on the N-terminal charge. From these observations, we propose that the function of N-terminal topogenic sequences depends on a balance between the N-terminal charge and the length of the following hydrophobic segment.
Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney.
Authors: Yamada Y , Post S R , Wang K , Tager H S , Bell G I , Seino S
Published: May 2006

Somatostatin is a tetradecapeptide that is widely distributed in the body. It acts on multiple organs including brain, pituitary, gut, exocrine and endocrine pancreas, adrenals, thyroid, and kidneys to inhibit release of many hormones and other secretory proteins. In addition, it functions as a neuropeptide affecting the electrical activity of neurons. Somatostatin exerts its biological effects by binding to specific high-affinity receptors, which appear in many cases to be coupled to GTP-binding proteins. Here we report the cloning, functional expression, and tissue distribution of two different somatostatin receptors (SSTRs). SSTR1 and SSTR2 contain 391 and 369 amino acids, respectively, and are members of the superfamily of receptors having seven transmembrane segments. There is 46% identity and 70% similarity between the amino acid sequences of SSTR1 and SSTR2. Stably transfected Chinese hamster ovary cells expressing SSTR1 or SSTR2 exhibit specific somatostatin binding, with an apparently higher affinity for somatostatin-14 than somatostatin-28, and NH2-terminally extended form of somatostatin-14. RNA blotting studies show that SSTR1 and SSTR2 are expressed at highest levels in jejunum and stomach and in cerebrum and kidney, respectively. A SSTR1 probe hybridized to multiple DNA fragments in EcoRI digests of human and mouse DNA, indicating that SSTR1 and SSTR2 are members of a larger family of somatostatin receptors. Thus, the biological effects of somatostatin are mediated by a family of receptors that are expressed in a tissue-specific manner.
Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini.
Authors: Silber K R , Keiler K C , Sauer R T
Published: May 2006

An Escherichia coli protease designated Tsp (tail-specific protease) has been purified, and its gene has been cloned and sequenced. Tsp specifically degrades a variant of the N-terminal domain of lambda repressor in which the five C-terminal residues, which are polar in wild type, have been replaced by nonpolar residues. This substrate specificity in vitro parallels the previously reported selective degradation in vivo of N-terminal-domain variants with nonpolar C-terminal residues. The gene sequence and N-terminal protein sequence of Tsp predict a protein of 660 amino acids. The deduced protein sequence of Tsp shows no significant homology to known protease sequences but does show sequence similarity to the human and bovine interphotoreceptor retinoid-binding proteins, which bind hydrophobic ligands.
Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor beta after antigen-specific triggering.
Authors: Miller A , Lider O , Roberts A B , Sporn M B , Weiner H L
Published: May 2006

Oral administration of myelin basic protein (MBP) is an effective way of suppressing experimental autoimmune encephalomyelitis (EAE). We have previously shown that such suppression is mediated by CD8+ T cells, which adoptively transfer protection and suppress immune responses in vitro. In the present study we have found that modulator cells from animals orally tolerized to MBP produce a suppressor factor upon stimulation with MBP in vitro that is specifically inhibited by anti-transforming growth factor beta (TGF-beta) neutralizing antibodies. No effect was observed with antibodies to gamma interferon, tumor necrosis factor alpha/beta, or indomethacin. In addition, the active form of the type 1 isoform of TGF-beta 1 (TGF-beta 1) can be directly demonstrated in the supernatants of cells from animals orally tolerized to MBP or ovalbumin after antigen stimulation in vitro. Antiserum specific for TGF-beta 1 administered in vivo abrogated the protective effect of oral tolerization to MBP in EAE. Furthermore, injection of anti-TGF-beta 1 serum to nontolerized EAE animals resulted in an increase in severity and duration of disease. These results suggest that immunomodulation of EAE induced by oral tolerization to MBP and natural recovery mechanisms use a common immunoregulatory pathway that is dependent on TGF-beta 1. Implications of such an association are of therapeutic relevance to human autoimmune diseases and may help to explain one of the mechanisms involved in the mediation of active suppression by T cells.
Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.
Authors: Aoki D , Lee N , Yamaguchi N , Dubois C , Fukuda M N
Published: May 2006

Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and that particularly Cys29 and His32 in this region are critical for GT to be retained in the Golgi.
Primary structure of high molecular weight tau present in the peripheral nervous system.
Authors: Couchie D , Mavilia C , Georgieff I S , Liem R K , Shelanski M L , Nunez J
Published: May 2006

The tau proteins are a family of brain microtubule binding proteins that are required during axonal outgrowth and are found in neurofibrillary tangles in Alzheimer disease. A protein of higher molecular weight, immunologically related to tau, is expressed in the adult peripheral system and in cultured neuronal cell lines of neural crest origin. The predicted amino acid sequence of the high molecular weight tau from N115 cells has been determined from the sequence of its 2340-base-pair cDNA. High molecular weight tau contains an open reading frame encoding 733 amino acid residues. It contains sequences homologous to those present in the N-, middle, and C-terminal domains of adult brain tau proteins, including four homologous repeats, which are the tubulin binding sites, and an amino acid stretch, which is present only in the N-terminal domain of the mature brain variants. The middle region contains a previously unidentified nonhomologous stretch of 237 amino acid residues as well as a domain of 66 residues homologous to exon 6 of the bovine gene that is absent in all bovine, rat, and mouse tau cDNAs sequenced so far. A cDNA probe specific to the nonhomologous tau insert hybridizes to the 8- to 9-kilobase tau mRNA in N115 cells but not to the 6-kilobase tau mRNA in brain. Probes for the domains common to brain tau isoforms hybridize to both messages. The sequence of high molecular weight tau protein also suggests that it, like low molecular weight tau, is an elongated hydrophilic molecule. This cDNA should allow us to study the role of the domains specific to these tau forms in the specialization of the peripheral nervous system and for study of their expression in normal and pathological states.
A leucine-to-proline mutation in the putative first transmembrane domain of the 22-kDa peripheral myelin protein in the trembler-J mouse.
Authors: Suter U , Moskow J J , Welcher A A , Snipes G J , Kosaras B , Sidman R L , Buchberg A M , Shooter E M
Published: May 2006

Peripheral myelin protein PMP-22 is a potential growth-regulating myelin protein that is expressed by Schwann cells and predominantly localized in compact peripheral myelin. A point mutation in the Pmp-22 gene of inbred trembler (Tr) mice was identified and proposed to be responsible for the Tr phenotype, which is characterized by paralysis of the limbs as well as tremors and transient seizures. In support of this hypothesis, we now report the fine mapping of the Pmp-22 gene to the immediate vicinity of the Tr locus on mouse chromosome 11. Furthermore, we have found a second point mutation in the Pmp-22 gene of trembler-J (TrJ) mice, which results in the substitution of a leucine residue by a proline residue in the putative first transmembrane region of the PMP-22 polypeptide. Tr and TrJ were previously mapped genetically as possible allelic mutations giving rise to similar, but not identical, phenotypes. This finding is consistent with the discovery of two different mutations in physicochemically similar domains of the PMP-22 protein. Our results strengthen the hypothesis that mutations in the Pmp-22 gene can lead to heterogeneous forms of peripheral neuropathies and offer clues toward possible explanations for the dominant inheritance of these disorders.
Cloning of the rat gene encoding choline acetyltransferase, a cholinergic neuron-specific marker.
Authors: Hahn M , Hahn S L , Stone D M , Joh T H
Published: May 2006

The neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT; EC 2.3.1.6). Since the expression of ChAT in the nervous system is restricted to cholinergic neurons, it serves as a specific marker for these neurons. In Alzheimer disease, ChAT activity is markedly reduced in the affected brain areas. Nerve growth factor can increase the ChAT activity of brain cholinergic neurons in vitro and in vivo. We have cloned the rat ChAT gene and identified one 5' noncoding exon and 14 exons that account for the entire coding sequence. The exon organization is compared with the protein domains conserved during evolution. These exons are distributed over at least 64 kilobases in the rat genome; the largest intron is at least 14 kilobases long. Within a 0.7-kilobase region immediately upstream of the confirmed sequence of the noncoding exon, TATA-like elements and numerous potential binding sites for transcription factors are found, including AP-1, Sp1, octamer-binding factor(s), CTF/NF-1, and the nuclear oncoprotein Myb.
On the structure of the nickel/iron/sulfur center of the carbon monoxide dehydrogenase from Rhodospirillum rubrum: an x-ray absorption spectroscopy study.
Authors: Tan G O , Ensign S A , Ciurli S , Scott M J , Hedman B , Holm R H , Ludden P W , Korszun Z R , Stephens P J , Hodgson K O
Published: May 2006

The nickel/iron/sulfur center of the carbon monoxide dehydrogenase (carbon monoxide:(acceptor)oxidoreductase; EC 1.2.99.2) enzyme from Rhodospirillum rubrum (Rr-CODH) was studied by x-ray absorption spectroscopy at the Ni K edge. Extended x-ray absorption fine structure data show that the first Ni coordination shell consists of 2 S atoms at 2.23 A and 2-3 N/O atoms at 1.87 A. The edge structure indicates a distorted tetrahedral or five-coordinate Ni environment in both oxidized and reduced Rr-CODH. By comparing second-shell extended x-ray absorption fine structure data of Rr-CODH to that of (Et4N)3[NiFe3S4(SEt)4], a cubane-type cluster, it was clearly established that Ni in the Rr-CODH center is not involved in the core of a NiFe3S4 cubane cluster. One model consistent with the results is a mononuclear Ni2+ site, bridged by S-Cys or sulfide to one or both of the Fe4S4 clusters of the enzyme, with the remaining coordination sites occupied by additional S-Cys or N/O-liganding amino acid residues.
Environment affects amino acid preference for secondary structure.
Authors: Zhong L , Johnson W C
Published: May 2006

Three equivocal amino acid sequences were synthesized that are predicted to be alpha-helical from amino acid preference but are found to be primarily beta-strand from x-ray diffraction of their respective proteins. In some solvent systems we recover the alpha-helical structure predicted by amino acid preference, whereas in other systems we mimic the interior of the protein and produce a beta-strand. These results are experimental proof that the environment is important in determining the secondary structure formed by an amino acid sequence; therefore schemes that predict secondary structure from amino acid sequence alone can never be totally successful.
Methods for the large-scale synthesis of psoralen furan-side monoadducts and diadducts.
Authors: Spielmann H P , Sastry S S , Hearst J E
Published: May 2006

We report methods for the preparation of multimicromole quantities of extremely pure uniquely photo-adducted psoralen-DNA furan-side monoadducts and diadducts (cross-links). The methods use high-intensity krypton and argon ion lasers in the photoreactions and HPLC methods to purify the required oligonucleotides containing the photoadducts. With these methods we have synthesized 2-3 mumol of 8-mer psoralen furan-side monoadduct and diadduct. These methods allow one to generate large amounts of psoralenated DNA oligonucleotides and facilitate their study by NMR and x-ray crystallography.
Modification of histidine residues in proteins by reaction with 4-hydroxynonenal.
Authors: Uchida K , Stadtman E R
Published: May 2006

We find that histidine residues in proteins are major targets for reaction with the lipid peroxidation product 4-hydroxynon-2-enal (HNE). Reaction of insulin (which contains no sulfhydryl groups) with HNE leads to the generation of HNE-protein adducts, which are converted to radioactive derivatives upon subsequent treatment with NaB[3H]H4. Amino acid analysis of the modified protein showed that the HNE treatment leads to the selective loss of histidine residues and the stiochiometric formation of 3H-labeled amino acid derivatives. The same labeled products were detected in acid hydrolysates of polyhistidine and N-acetylhistidine after their reactions with HNE and NaB[3H]H4. The reaction of N-acetylhistidine with HNE led to the production of two compounds. Upon acid hydrolysis, both derivatives yielded stoichiometric amounts of histidine. However, after reduction with NaBH4, acid hydrolysis led to a mixture of amino acid derivatives [presumably, isomeric forms of N pi (N tau)-1,4-dihydroxynonanylhistidine] that were indistinguishable from those obtained from insulin and polyhistidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by HNE involves a Michael-type addition of the imidazole nitrogen atom of histidine to the alpha, beta-unsaturated bond of HNE, followed by secondary reaction involving the aldehyde group with the C-4 hydroxyl group of HNE. The reaction of histidine residues with HNE provides the basis for methods by which the contributions of HNE in the modification of proteins can be determined.
Estrogen receptors colocalize with low-affinity nerve growth factor receptors in cholinergic neurons of the basal forebrain.
Authors: Toran-Allerand C D , Miranda R C , Bentham W D , Sohrabji F , Brown T J , Hochberg R B , MacLusky N J
Published: May 2006

The rodent and primate basal forebrain is a target of a family of endogenous peptide signaling molecules, the neurotrophins--nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3--and of the gonadal steroid hormone estrogen, both of which have been implicated in cholinergic function. To investigate whether or not these ligands may act on the same neurons in the developing and adult rodent basal forebrain, we combined autoradiography with 125I-labeled estrogen and either nonisotopic in situ hybridization histochemistry or immunohistochemistry. We now report colocalization of intranuclear estrogen binding sites with the mRNA and immunoreactive protein for the low-affinity nerve growth factor receptor, which binds all three neurotrophins, and for the cholinergic marker enzyme choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). Colocalization of estrogen and low-affinity nerve growth factor receptors implies that their ligands may act on the same neuron, perhaps synergistically, to regulate the expression of specific genes or gene networks that may influence neuronal survival, differentiation, regeneration, and plasticity. That cholinergic neurons in brain regions subserving cognitive functions may be regulated not only by the neurotrophins but also by estrogen may have considerable relevance for the development and maintenance of neural substrates of cognition. If estrogen-neurotrophin interactions are important for survival of target neurons, then clinical conditions associated with estrogen deficiency could contribute to the atrophy or death of these neurons. These findings have implications for the subsequent decline in those differentiated neural functions associated with aging and Alzheimer disease.
Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells.
Authors: Sanders M C , Goldstein A L , Wang Y L
Published: May 2006

Thymosin beta 4 (beta 4) is a 5-kDa polypeptide originally identified in calf thymus. Although numerous activities have been attributed to beta 4, its physiological role remains elusive. Recently, beta 4 was found to bind actin in human platelet extracts and to inhibit actin polymerization in vitro, raising the possibility that it may be a physiological regulator of actin assembly. To examine this potential function, we have increased the cellular beta 4 concentration by microinjecting synthetic beta 4 into living epithelial cells and fibroblasts. The injection induced a diminution of stress fibers and a dose-dependent depolymerization of actin filaments as indicated by quantitative image analysis of phalloidin binding. Our results show that beta 4 is a potent regulator of actin assembly in living cells.
1 alpha,25-dihydroxyvitamin D3 regulates the transcription of carbonic anhydrase II mRNA in avian myelomonocytes.
Authors: Lomri A , Baron R
Published: May 2006

Carbonic anhydrase II (CAII) is highly expressed in the osteoclast, where it is involved in the process of extracellular acidification required for bone resorption. We have previously shown that 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], a steroid hormone that regulates the differentiation of macrophages and osteoclasts, induces the expression of CAII mRNA and protein in avian bone marrow cells. To determine whether this regulation occurred at the gene level, we have studied the effects of 1,25(OH)2D3 on CAII expression in a transformed myelomonocytic avian cell line (BM2). As observed in nontransformed cells, 1,25(OH)2D3 markedly increased CAII biosynthesis and mRNA levels. The increase in CAII mRNA was detected as early as 3 hr after adding the hormone (1.9-fold) and reached 4.7-fold by 48 hr. These effects were completely blocked by actinomycin D, and nuclear run-on analysis confirmed that 1,25(OH)2D3 increased the rate of CAII gene transcription. In contrast, induction of CAII mRNA expression was not affected by inhibition of protein synthesis with cycloheximide, and no significant changes in mRNA stability were seen. Thus, 1,25(OH)2D3 modulates CAII gene expression at the transcriptional level, and this effect does not require de novo synthesis of other gene products. These results suggest that activation of the CAII gene occurs early in the differentiation events triggered by vitamin D3 in myelomonocytic cells.
Zinc finger point mutations within the WT1 gene in Wilms tumor patients.
Authors: Little M H , Prosser J , Condie A , Smith P J , Van Heyningen V , Hastie N D
Published: May 2006

A proposed Wilms tumor gene, WT1, which encodes a zinc finger protein, has previously been isolated from human chromosome 11p13. Chemical mismatch cleavage analysis was used to identify point mutations in the zinc finger region of this gene in a series of 32 Wilms tumors. Two exonic single base changes were detected. In zinc finger 3 of a bilateral Wilms tumor patient, a constitutional de novo C----T base change was found changing an arginine to a stop codon. One tumor from this patient showed allele loss leading to 11p hemizygosity of the abnormal allele. In zinc finger 2 of a sporadic Wilms tumor patient, a C----T base change resulted in an arginine to cysteine amino acid change. To our knowledge, a WT1 gene missense mutation has not been detected previously in a Wilms tumor. By comparison with a recent NMR and x-ray crystallographic analysis of an analogous zinc finger gene, early growth response gene 1 (EGR1), this amino acid change in WT1 occurs at a residue predicted to be critical for DNA binding capacity and site specificity. The detection of one nonsense point mutation and one missense WT1 gene point mutation adds to the accumulating evidence implicating this gene in a proportion of Wilms tumor patients.
Expression pattern of the RAR alpha-PML fusion gene in acute promyelocytic leukemia.
Authors: Alcalay M , Zangrilli D , Fagioli M , Pandolfi P P , Mencarelli A , Lo Coco F , Biondi A , Grignani F , Pelicci P G
Published: May 2006

Two chimeric genes, PML-RAR alpha and RAR alpha-PML, are formed as a consequence of the acute promyelocytic leukemia (APL)-specific reciprocal translocation of chromosomes 15 and 17 [t(15;17)]. PML-RAR alpha is expressed as a fusion protein. We investigated the organization and expression pattern of the RAR alpha-PML gene in a series of APL patients representative of the molecular heterogeneity of the t(15;17) and found (i) two types of RAR alpha-PML mRNA junctions (RAR alpha exon 2/PML exon 4 or RAR alpha exon 2/PML exon 7) that maintain the RAR alpha and PML longest open reading frames aligned and are the result of chromosome 15 breaking at two different sites; and (ii) 10 different RAR alpha-PML fusion transcripts that differ for the assembly of their PML coding exons. A RAR alpha-PML transcript was present in most, but not all, APL patients.
Higher order interactions in 23s rRNA.
Authors: Larsen N
Published: May 2006

An alignment of 75 phylogenetically diverse large subunit ribosomal RNA sequences was created and searched for secondary and tertiary structure elements by computer. The search revealed four unknown secondary structural pairings, two internal loop closings, and five short-range tertiary interactions--two of which were pairings of an unusual type. One brings a loop together with two other loops previously known to be paired, and one involves a nucleotide within a presumed tetraloop. The latter interaction constrains the RNA structure near the ribosomal E-site, where two base pairs previously suggested to be in parallel orientation are now proven. No clear phylogenetic evidence for direct base pairing between the large and small subunit rRNA was found.
Transforming growth factor beta induces myoblast differentiation in the presence of mitogens.
Authors: Zentella A , Massagué J
Published: May 2006

Transforming growth factor beta 1 (TGF-beta 1) added to L6E9 rat skeletal myoblasts in mitogen-rich medium induces a rapid decrease in c-myc expression and delays progression through the G1 phase of the cell cycle. This growth inhibitory response is followed by cell commitment to terminal differentiation with elevated expression of myogenin muscle determination genes, induction of muscle-specific proteins, and formation of multinucleated myotubes. These results suggest that TGF-beta 1 may act as a physiological inducer of myogenic differentiation in mitogen-rich environments, and its otherwise reversible growth inhibitory effect may become permanent if coupled to induction of terminal differentiation.
Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages.
Authors: Cianciotto N P , Fields B S
Published: May 2006

Legionella pneumophila is an intracellular parasite of freshwater protozoa and human macrophages. Recent studies determined that the macrophage infectivity potentiator (Mip) surface protein, a prokaryotic homolog of the FK506-binding proteins, is required for optimal infection of macrophages. To determine whether Mip is also involved in L. pneumophila infection of protozoa, we examined the ability of a strain lacking Mip to parasitize Hartmannella amoebae and Tetrahymena ciliates. After 3 days of incubation, approximately 1000-fold fewer bacteria were recovered from protozoan cocultures infected with the Mip- strain than from those cocultures infected with an isogenic Mip+ strain. The mip mutant was, however, not impaired in its ability to bind to amoebae cell surfaces, indicating that Mip is involved in bacterial resistance to intracellular killing and/or intracellular multiplication. These data suggest that L. pneumophila employs similar genes and mechanisms to infect human cells and protozoa. Furthermore, they support the hypothesis that the ability of L. pneumophila to parasitize macrophages and hence to cause human disease is a consequence of its prior adaptation to intracellular growth within protozoa.
Direct effect of basic fibroblast growth factor on gene transcription in a cell-free system.
Authors: Nakanishi Y , Kihara K , Mizuno K , Masamune Y , Yoshitake Y , Nishikawa K
Published: May 2006

Recent findings on the translocation of intact fibroblast growth factor (FGF) into the cell nucleus suggest that it functions directly in nuclear events. We examined the effect of human basic FGF (bFGF) on gene transcription in a cell-free system. When mouse genes encoding phosphoglycerate kinases 1 and 2 (Pgk-1 and Pgk-2) were transcribed by using nuclear extracts of Ehrlich ascites tumor cells, FGF affected transcription in different ways: in the presence of bFGF, transcription of the Pgk-1 gene was inhibited, whereas that of the Pgk-2 gene was enhanced. When viral genes were tested, transcription of the adenovirus major late DNA was slightly stimulated but that of the adenovirus early E1A DNA or the human immunodeficiency virus DNA was not changed by the addition of bFGF. Moreover, the presence of a distinct 5' upstream region of the Pgk-2 gene, which includes a negative cis-acting element, was required for transcription stimulation by bFGF. These results suggest that bFGF can regulate transcription directly in the nucleus in a gene-specific manner.
Enumeration of lymphokine mRNA-containing cells in vivo in a murine graft-versus-host reaction using the PCR.
Authors: Troutt A B , Kelso A
Published: May 2006

A method of enumerating lymphokine mRNA-containing cells in vivo was developed by combining limiting dilution analysis with PCR amplification of cDNA. Single-hit kinetics revealed that the PCR-limiting dilution analysis could detect a single positive cell among greater than 40,000 negative cells. With this method, spleens and lymph nodes of mice undergoing an acute allogeneic graft-versus-host reaction were found to contain lymphokine mRNA-expressing cells at frequencies of 3% for interferon gamma, 0.05% for granulocyte/macrophage colony-stimulating factor, 0.002% for interleukin 3, and 0.03% for interleukin 4; these frequencies were 20- to 175-fold higher than in lymphoid tissues of normal mice. In contrast to their low frequencies of lymphokine mRNA-containing cells in vivo, graft-versus-host reaction populations restimulated in vitro for 24 hr with anti-CD3 antibody yielded frequencies ranging from 3% for interleukin 4 to nearly 70% for interferon gamma. Furthermore, lymphokine transcripts were also detected in single micromanipulated cells from these populations. Because frequencies of anti-CD3-inducible lymphokine mRNA-containing cells in normal mice were only 0.03-1%, it was concluded that lymphoid tissues of graft-versus-host reaction mice contained high frequencies of cells that had been primed for lymphokine synthesis. Only a small fraction of these cells, however, expressed lymphokine mRNAs at a given time point in vivo.
Genotypic analysis of N-ethyl-N-nitrosourea-induced mutations by Taq I restriction fragment length polymorphism/polymerase chain reaction in the c-H-ras1 gene.
Authors: Chiocca S M , Sandy M S , Cerutti P A
Published: May 2006

In genotypic mutation analysis DNA sequence changes are determined without the in vivo or in vitro selection of phenotypically altered cells. We have studied the induction of base-pair changes by N-ethyl-N-nitrosourea in Taq I endonuclease recognition site 2508-2511 (TCGA) of the c-H-ras1 gene in human fibroblasts by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) method. This site contains the four bases, and all 12 possible single base-pair changes can be monitored. The transition of guanine to adenine at position 2510 was the major mutation detected by lambda plaque oligonucleotide hybridization and quantitative sequence analysis of the RFLP/PCR products. It involves the G residue of the CpG sequence of the coding strand. Data calibration with an internal mutant standard indicates that absolute frequencies for this transition lie in the range of 4-12 x 10(-7). The present study documents the capacity of the RFLP/PCR approach to measure mutagen-induced base-pair changes in a specific gene sequence without the selection of a phenotypically altered cell.