bluuebunny/crossref_metadata_2025 huggingface dataset:
Fractionated nuclear extracts from hamster cells catalyze cell-free recombination at selective sequences between adenovirus DNA and a hamster preinsertion site.
Authors: Tatzelt J , Fechteler K , Langenbach P , Doerfler W
Published: May 2006

We have explored the mechanism of adenovirus type 12 (Ad12) DNA integration because of its importance for viral oncogenesis and as an example of insertional recombination. We have used a fractionated cell-free system from nuclear extracts of hamster cells and have partly purified nuclear proteins that could catalyze in vitro recombination. As recombination partners, the 20,880- to 24,049-nucleotide Pst I D fragment of Ad12 DNA and the hamster preinsertion sequence p7 from the Ad12-induced tumor CLAC1 have proven to recombine at higher frequencies than randomly selected adenoviral or cellular DNA sequences. A preinsertion sequence might carry elements essential in eliciting recombination. Patch homologies between the recombination partners seem to play a role in the selection of sites for recombination in vivo and in the cell-free system. Nuclear extracts from BHK21 cells were prepared by incubating the nuclei in 0.42 M (NH4)2SO4 and fractionated by Sephacryl S-300 gel filtration, followed by chromatography on Mono S and Mono Q columns. The purified products active in recombination contained a limited number of different protein bands, as determined by polyacrylamide gel electrophoresis and silver staining. The most highly purified fraction IV had helicase and topoisomerase I activities. We used two different methods to assess the in vitro generation of hamster DNA-Ad12 DNA recombinants upon incubation with the purified protein fractions: (i) transfection of the recombination products into recA- strains of Escherichia coli and (ii) the polymerase chain reaction by using amplification primers unique for each of the two recombination partners. In p7 hamster DNA, the nucleotide sequence 5'-CCTCTCCG-3' or similar sequences served repeatedly as a preferred recombination target for Ad12 DNA in the tumor CLAC1 and in five independent cell-free recombination experiments.
Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid.
Authors: Mueller M J , Brodschelm W , Spannagl E , Zenk M H
Published: May 2006

Fungal cell walls and fragments thereof (elicitors) induce the formation of low and high molecular weight defense compounds in plant cell suspension cultures. This induced synthesis requires a signal molecule transmitting the message between the elicitor plant cell wall receptor and gene activation. We demonstrate in this study that cis-jasmonic acid is rapidly synthesized in plant cell cultures of diverse taxonomic origin (gymnosperms and mono- and dicotyledonous plants) after challenge with a fungal elicitor preparation. The rapid decline of cis-jasmonic acid in some of these tissues is attributed to rapid metabolism of this pentacyclic acid. The induction of alkaloids by several different molecules provoking the elicitation process is strictly correlated with the synthesis of jasmonates. Elicitation leads to a rapid release of alpha-linolenic acid from the lipid pool of the plant cell. alpha-Linolenic acid and 12-oxophytodienoic acid, the formation of which is also induced, are known to be distant precursors of jasmonic acid. We assume cis-jasmonic acid and its precursors to be the signaling molecules in the elicitation process.
Facilitated distortion of the DNA site enhances EcoRI endonuclease-DNA recognition.
Authors: Lesser D R , Kurpiewski M R , Waters T , Connolly B A , Jen-Jacobson L
Published: May 2006

We have measured the binding of EcoRI endonuclease to a complete set of purine-base analogue sites, each of which deletes one functional group that forms a hydrogen bond with the endonuclease in the canonical GAATTC complex. For five of six functional group deletions, the observed penalty in binding free energy is +1.3 to +1.7 kcal/mol. For two of these cases (replacement of adenine N7 with carbon) a single protein-base hydrogen bond is removed without deleting an interstrand Watson-Crick hydrogen bond or causing structural "adaptation" in the complex. This observation establishes that the incremental energetic contribution of one protein-base hydrogen bond is about -1.5 kcal/mol. By contrast, deletion of the N6-amino group of the inner adenine in the site improves binding by -1.0 kcal/mol because the penalty for deleting a protein-base hydrogen bond is outweighed by facilitation of the required DNA distortion ("kinking") in the complex. This result provides direct evidence that the energetic cost of distorting a DNA site can make an unfavorable contribution to protein-DNA binding.
A cytoplasmic 57-kDa protein that is required for translation of picornavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein.
Authors: Hellen C U , Witherell G W , Schmid M , Shin S H , Pestova T V , Gil A , Wimmer E
Published: May 2006

Initiation of translation of the RNA genomes of picornaviruses such as poliovirus and encephalomyocarditis virus is cap-independent and results from interaction of ribosomes with a segment of the 5' noncoding region of these mRNAs termed the internal ribosomal entry site. Genetic and biochemical studies have previously shown that a 57-kDa cytoplasmic RNA-binding protein (p57) plays an essential role in this translation mechanism. We have now found that p57 shares physical, biochemical, and antigenic properties with the pyrimidine tract-binding protein (PTB), a nuclear protein that has been implicated in various processes involving pre-mRNA. These data indicate that p57 and PTB are the same protein. Purified recombinant PTB bound specifically to a bulged hairpin within the internal ribosomal entry site of encephalomyocarditis virus and had a much lower affinity for a mutated derivative of this hairpin and for unrelated RNAs. Immunodepletion of p57/PTB from a HeLa cell-free lysate inhibited translation of poliovirus and encephalomyocarditis virus mRNAs but had no effect on translation of beta-globin mRNA, confirming the essential role of p57 in translation by internal ribosomal entry.
Molecular regulation of human interleukin 2 and T-cell function by interleukin 4.
Authors: Schwarz E M , Salgame P , Bloom B R
Published: May 2006

Distinct functional T-cell subsets, differing in the patterns of lymphokines produced, regulate cell-mediated and humoral immune responses. The two major types and their principal products, interleukin 4 and interferon gamma (IL-4 and IFN-gamma), are reciprocally negatively interactive. To analyze the molecular mechanism of IL-4-mediated suppression of cell-mediated immunity we studied its effects on expression of interleukin 2 (IL-2) and IFN-gamma. IL-4 pretreatment of Jurkat cells prior to stimulation resulted in a decrease in transcription of the IL2 gene. IL-4 suppressed IL-2 and IFN-gamma mRNA levels in primary human T cells, and addition of anti-CD28 antibodies relieved this suppression. Using enhancer-reporter constructs, IL-4 specifically down-regulated the NFIL-2B element. Electrophoretic mobility shift assays using a DNA oligomer containing the NFIL-2B binding site indicated that IL-4 inhibited the NFIL-2B complex and that the NFIL-2B DNA binding factor is distinct from AP-1. These results suggest that IL-4 may regulate development and function of T-cell subsets involved in cell-mediated immunity in part by inhibiting factors required for transcription of the IL2 gene.
N-Methyl-D-aspartate receptors are clustered and immobilized on dendrites of living cortical neurons.
Authors: Benke T A , Jones O T , Collingridge G L , Angelides K J
Published: May 2006

The response of nerve cells to synaptic inputs and the propagation of this activation is critically dependent on the cell-surface distribution of ion channels. In the hippocampus, Ca2+ influx through N-methyl-D-aspartate receptors (NMDAR) and/or voltage-dependent calcium channels on dendrites is thought to be critically involved in long-term potentiation, neurite outgrowth, epileptogenesis, synaptogenesis, and cell death. We report that conantokin-G (CntxG), a peptide from Conus geographus venom, competitively blocked with high affinity and specificity NMDAR-mediated currents in hippocampal neurons and is a reliable probe for exploring NMDAR distribution. Fluorescent derivatives of CntxG were prepared and used to directly determine NMDAR distribution on living hippocampal neurons by digital imaging and confocal fluorescence microscopy. In hippocampal slices, the CA1 dendritic subfield was strongly labeled by CntxG, whereas the CA3 mossy fiber region was not. On CA1 hippocampal neurons in culture, dendritic CntkG-sensitive NMDAR were clustered at sites of synaptic contacts, whereas somatic NMDAR were distributed diffusely and in patches. NMDAR distribution differed from the distribution of voltage-dependent calcium channels. A significant fraction of labeled NMDAR on somata and dendrites was found to be highly mobile: rates were consistent with the possible rapid recruitment of NMDAR to specific synaptic locations. The localization of NMDAR and modulation of this distribution demonstrated here may have important implications for the events that underlie neuronal processing and synaptic remodeling during associative synaptic modification.
Expression of Chinese hamster cAMP-dependent protein kinase in Escherichia coli results in growth inhibition of bacterial cells: a model system for the rapid screening of mutant type I regulatory subunits.
Authors: Gosse M E , Padmanabhan A , Fleischmann R D , Gottesman M M
Published: May 2006

The regulatory and catalytic subunits of cAMP-dependent protein kinase (PKA) were coexpressed within the same bacterial cell using a polycistronic bacterial T7 expression vector encoding Chinese hamster cDNAs for the type I regulatory (RI) and catalytic alpha (C alpha) subunits of PKA. Basal expression of active RI/C alpha holoenzyme in the BL21(DE3) strain of Escherichia coli caused severe growth inhibition resulting in extremely small colony size. Several lines of evidence demonstrate that this growth inhibition requires active PKA subunits and cAMP: (i) this phenotype is dependent on cAMP since it is not seen in a strain lacking adenylyl cyclase activity, but the growth rate of these transformants is slower when exogenous cAMP is added; (ii) normal growth occurs when wild-type RI cDNA is replaced by a mutant RI cDNA encoding a RI protein with reduced cAMP binding; and (iii) the growth-inhibited phenotype of the transformed BL21(DE3) cells requires soluble, active C alpha protein. Holoenzyme expressed in bacteria is activated by cAMP, which stimulates phosphorylation of an endogenous 50-kDa protein that is missing in four host mutants selected for normal growth after transformation with PKA holoenzyme. A mutant RI cDNA library was generated by PCR random mutagenesis and screened by polycistronic expression in BL21(DE3) cells. The RI cDNA sequence from one revertant has base-pair substitutions creating two amino acid substitutions within the cAMP binding sites. The coexpression of the RI/C alpha subunits in BL21(DE3) bacterial cells provides a system for rapidly selecting mutations in the RI subunits of PKA.
Chicken repeat 1 elements contain a pol-like open reading frame and belong to the non-long terminal repeat class of retrotransposons.
Authors: Burch J B , Davis D L , Haas N B
Published: May 2006

Chicken genomes contain approximately 30,000 chicken repeat 1 (CR1) elements scattered among single-copy sequences, but no information has yet been presented to account for how these elements could have dispersed. The fact that CR1 elements have common (although atypical) 3' ends and variable 5' truncations suggested to us that they might belong to the class of non-long terminal repeat retrotransposons that encode reverse transcriptases. From an analysis of unusually large CR1 elements, we now provide evidence for the presence of such a reverse transcriptase open reading frame. CR1 elements are distantly related to previously described non-long terminal repeat retrotransposons; however, we find that frog and torpedo ray genomes contain dispersed open reading frame segments that have > 50% identity to the CR1 open reading frame. This result suggests that CR1-like elements exist in several vertebrate classes that have evolved independently for approximately 400 million years.
Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9.
Authors: Day K P , Karamalis F , Thompson J , Barnes D A , Peterson C , Brown H , Brown G V , Kemp D J
Published: May 2006

Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria.
Stereocilia displacement induced somatic motility of cochlear outer hair cells.
Authors: Evans B N , Dallos P
Published: May 2006

Outer hair cells, isolated from mammalian cochleas, are known to respond to electrical stimulation with elongation or contraction of the cell's cylindrical soma. It is assumed that such shape changes, when driven by the cell's receptor potential in vivo, are a part of the feedback process that underlies cochlear amplification. To date it has not been possible to demonstrate somatic shape changes upon normal mechanical stimulation of the cell--i.e., the deflection of its hair bundle. We show here that mechanically induced hair-bundle deflection produces somatic motility of the cell. Such motility is dependent upon a functioning forward transducer process and disappears upon interference with transduction. The motile response also reflects the hair bundle's known directional sensitivity. This demonstration of mechanically driven motility indicates that the cell may possess capabilities to affect its mechanical environment under control of its own receptor potential and, thereby, participate in a local cochlear feedback process.
Polyunsaturated fatty acids inhibit S14 gene transcription in rat liver and cultured hepatocytes.
Authors: Jump D B , Clarke S D , MacDougald O , Thelen A
Published: May 2006

Polyunsaturated fatty acids (PUFAs) have been shown to have significant effects on hepatic lipogenic gene expression. The S14 gene has been used as a model to examine the effects of PUFAs on hepatic lipogenic gene expression. In vivo studies showed that feeding rats a high carbohydrate diet containing menhaden oil rapidly (within hours) and significantly (> or = 50%) attenuates hepatic S14 gene transcription and S14 mRNA abundance. The suppressive effect of menhaden oil was both gene and tissue specific. The effect of PUFAs on expression of the S14 mRNA and a transfected S14 fusion gene (i.e., S14CAT4.3) was examined in cultured hepatocytes in the presence of triiodothyronine (T3), insulin, dexamethasone, and albumin under serum-free conditions. Whereas T3 stimulated both S14 mRNA (> 40-fold) and S14CAT4.3 (> 100-fold), eicosapentaenoic acid (C20:5 omega 3) significantly attenuated (> or = 80%) both S14 mRNA and S14CAT activity in a dose-dependent fashion. The effects of C20:5 on hepatocyte gene expression were both gene and fatty acid specific. Deletion analysis of transfected S14CAT fusion genes indicated that the S14 thyroid hormone response element (at -2.5 to -2.9 kb) was not sensitive to C20:5 control. The cis-linked PUFA response elements were localized to a region within the S14 proximal promoter (at -80 to -220 bp). This region also contains cis-acting elements that potentiate T3 activation of S14 gene transcription. These studies suggest that C20:5 (or its metabolites) regulates factors within the S14 proximal promoter region that are important for T3 activation of S14 gene transcription.
Decatenation activity of topoisomerase IV during oriC and pBR322 DNA replication in vitro.
Authors: Peng H , Marians K J
Published: May 2006

Topoisomerase IV (Topo IV), encoded by parC and parE, is required for partition of the daughter chromosomes in Escherichia coli. This enzyme is likely responsible for decatenating the linked daughter chromosomes after replication. In this report, we have examined the action of Topo IV in both pBR322 and oriC DNA replication reconstituted in vitro with purified proteins. Gyrase fails to decatenate the linked daughter molecules under any condition in the oriC system and at physiological salt concentrations in the pBR322 system, whereas Topo IV stimulates generation of monomer product DNA by 7- to 10-fold. Topo IV-catalyzed decatenation of isolated multiply linked DNA dimers was relatively insensitive to salt; it proceeded at 14% of the maximal rate even in the presence of 800 mM potassium glutamate. In contrast, decatenation in vitro by gyrase was inhibited completely under these conditions. Pulse-chase analysis indicated that Topo IV-catalyzed resolution of linked daughter DNA molecules occurred prior to completion of DNA replication, such that multiply linked daughter molecules did not arise. These results suggest that during DNA replication, gyrase acts primarily to relieve accumulated positive supercoiling and Topo IV acts to segregate the daughter chromosomes.
Expression cloning of a human corticotropin-releasing-factor receptor.
Authors: Chen R , Lewis K A , Perrin M H , Vale W W
Published: May 2006

Corticotropin-releasing factor (CRF) is the principal neuroregulator of the hypothalamic-pituitary-adrenocortical axis and plays an important role in coordinating the endocrine, autonomic, and behavioral responses to stress and immune challenge. We report here the cloning of a cDNA coding for a CRF receptor from a human corticotropic tumor library. The cloned cDNA encodes a 415-amino acid protein comprising seven putative membrane-spanning domains and is structurally related to the calcitonin/vasoactive intestinal peptide/growth hormone-releasing hormone subfamily of G protein-coupled receptors. The receptor expressed in COS cells binds rat/human CRF with high affinity (Kd = 3.3 +/- 0.45 nM) and specificity and is functionally coupled to adenylate cyclase. The CRF antagonist alpha-helCRF-(9-41) inhibits the CRF-stimulated increase in intracellular cAMP. Northern blot analysis reveals that the CRF receptor is expressed in the rat pituitary and brain as well as in the mouse AtT20 corticotropic cells. We also describe an alternatively spliced form of the receptor which includes an insert of 29 amino acids in the first intracellular loop.
Increasing antigenic and genetic diversity of the V3 variable domain of the human immunodeficiency virus envelope protein in the course of the AIDS epidemic.
Authors: Kuiken C L , Zwart G , Baan E , Coutinho R A , van den Hoek J A , Goudsmit J
Published: May 2006

Population-wide variation in genomic RNA of human immunodeficiency virus type 1 (HIV-1) encompassing the V3 loop of the envelope protein was studied in serum samples of 74 newly infected individuals from three Dutch cohorts: 30 homosexual men, 32 drug users, and 12 hemophiliacs. During acute infection, HIV-1 RNA sequences present in serum are relatively homogeneous, which makes direct sequencing feasible. This offered an opportunity to study the infecting virus variants before mutations had accumulated in the new host. The sampling dates ranged from 1980 to 1991, thus spanning the entire AIDS epidemic in The Netherlands. The diversity in the sequenced region increased over time in both the homosexual and the drug-user risk groups. Furthermore, this increase was associated with an increase in antigenic variation, as witnessed by serum reactivity to a V3 peptide panel. Despite this diversification, some 1990 sequences still closely resembled the earliest 1980 sequence, making ancestral inferences problematic. No evidence was found of a change in the master sequence of the virus quasi-species over time. At the amino acid level, no risk-group-associated variation was found, but at the nucleotide level, the drug-user and homosexual/hemophiliac sequences could be distinguished on the basis of a single silent nucleotide change in the sequence encoding the tip of the V3 loop. Hemophiliac sequences could not be distinguished from those of homosexuals. In spite of the large and increasing genetic variability, all sequences were more similar to the European/American HIV consensus sequence than to that of non-Western strains.
Platelet-derived growth factor (PDGF) in oncogenesis: development of a vascular connective tissue stroma in xenotransplanted human melanoma producing PDGF-BB.
Authors: Forsberg K , Valyi-Nagy I , Heldin C H , Herlyn M , Westermark B
Published: May 2006

Human WM9 melanoma cells, previously shown to be devoid of PDGF expression, were stably transfected with a PDGF-B cDNA under the transcriptional control of a cytomegalovirus promoter. Northern blot analysis revealed high expression of an mRNA of the expected size in the PDGF-B-transfected cells. Synthesis and secretion of PDGF-BB was confirmed by immunoprecipitation. Furthermore, conditioned medium from PDGF-B-transfected cells contained a mitogenic activity for fibroblasts. For analysis of tumor growth in vivo, cells of each type were injected subcutaneously into BALB/c nu/nu mice. Tumors from mice injected with WM9 cells transfected with the vector only contained large necrotic areas; only scant blood vessels with narrow lumina were observed. No connective tissue was present. In the tumors from PDGF-B-transfected WM9 cells, nests of tumor were divided by connective tissue septa. An abundance of blood vessels was observed in the connective tissue septa and within the tumor cell nests. There was a complete absence of necrosis in these tumors. The present results suggest that tumor-derived PDGF-BB is a potent mediator of connective tissue stroma formation. The connective tissue framework that is generated in response to PDGF-BB may form a solid support for newly formed blood vessels and, thereby, facilitate the formation of a functional vascular system in the tumor.
Insulin down-regulates expression of the insulin-responsive glucose transporter (GLUT4) gene: effects on transcription and mRNA turnover.
Authors: Flores-Riveros J R , McLenithan J C , Ezaki O , Lane M D
Published: May 2006

Insulin rapidly represses expression of the gene encoding the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 mouse adipocytes. Upon exposure to the hormone the cellular level of GLUT4 mRNA falls (t1/2 approximately 2.5 hr) to 20-30% of its initial level within 10 hr. This is followed by a similar decrease in the level of GLUT4 protein. Down-regulation of GLUT4 mRNA is a result of both rapid repression of transcription of the GLUT4 gene and an increased rate of turnover of the GLUT4 message. As a consequence of prolonged exposure to insulin, 3T3-L1 adipocytes lose their capacity for acute stimulation of hexose uptake by insulin. These findings provide an explanation for the resistance of glucose uptake to insulin in adipose tissue observed in non-insulin-dependent (type 2) diabetes mellitus, particularly that associated with hyperinsulinemia and obesity.
Identification of an RNA binding site for human thymidylate synthase.
Authors: Chu E , Voeller D , Koeller D M , Drake J C , Takimoto C H , Maley G F , Maley F , Allegra C J
Published: May 2006

Previous studies from this laboratory have shown that human TS mRNA translation is regulated by its protein product in a negative autoregulatory manner. In this paper, we identify an RNA binding site for TS protein located within the first 188 nt of TS RNA. A 36-nt RNA sequence contained within this 188-nt fragment, corresponding to nt 75-110 and including the translational initiation site, binds TS protein with an affinity similar to that of both the full-length and the 188-nt TS RNA sequences. Variant RNAs with either a deletion or a mutation at the translational initiation region are unable to compete for TS protein binding. UV crosslinking studies reveal that an RNA fragment of approximately 36 nt is protected from RNase T1 digestion by TS protein binding. A second TS protein-binding site is localized within the protein-coding region corresponding to nt 434-634. These findings demonstrate a specific interaction between human TS protein and its TS RNA and identify an RNA binding site that includes the translational initiation site.
A cyclophilin-related protein involved in the function of natural killer cells.
Authors: Anderson S K , Gallinger S , Roder J , Frey J , Young H A , Ortaldo J R
Published: May 2006

Natural killer cells are non-major histocompatibility complex-restricted large granular lymphocytes that can recognize and destroy tumor cells without prior stimulation. A 150-kDa molecule on the surface of human natural killer cells was identified as a component of a putative tumor-recognition complex. We report here the isolation of cDNAs coding for the 150-kDa tumor-recognition molecule from human and mouse cDNA libraries. The amino terminus of the predicted protein contains a large hydrophobic region followed by a domain that is highly homologous to cyclophilin/peptidylprolyl cis-trans isomerase. The remainder of the protein is extremely hydrophilic and contains three homologous positively charged clusters. There are also three regions that contain extensive arginine- and serine-rich repeats. Comparison of the human and mouse predicted amino acid sequences revealed > 80% homology.
A stochastic interpretation of the Riemann zeta function.
Authors: Alexander K S , Baclawski K , Rota G C
Published: May 2006

We give a stochastic process for which the terms of the Riemann zeta function occur as the probability distributions of the elementary random variables of the process.
Twist constraints on linker DNA in the 30-nm chromatin fiber: implications for nucleosome phasing.
Authors: Yao J , Lowary P T , Widom J
Published: May 2006

Previous work has shown that nucleosome repeat lengths, and hence linker DNA lengths, are preferentially quantized to a set of values differing by integral multiples of the helical twist of DNA. An explanation was proposed in which this preferential quantitation is due to twist constraints on linker DNA arising from nucleosome-nucleosome interactions in folded chromatin. Here we report the results of a study, using ethidium intercalation, designed to test whether twist constraints do indeed exist. Electron microscopy reveals that ethidium intercalation causes decondensation of dinucleosomes. Direct measurement of the free energy of intercalation by fluorescence spectroscopy reveals competition between chromatin folding and ethidium intercalation. Results from other laboratories establish that these effects of ethidium are due to ethidium-induced changes in the twist of linker DNA, and not to a variety of other possible effects. We conclude that twist constraints on linker DNA do exist. These may explain the observation of preferentially quantized linker DNA lengths. Implications of these results for mechanisms of nucleosome phasing and the mechanisms of drug action are discussed.
Peripheralization of hemopoietic progenitors in primates treated with anti-VLA4 integrin.
Authors: Papayannopoulou T , Nakamoto B
Published: May 2006

Interaction of hemopoietic cells with the elements of the underlying bone marrow stroma, the unique site of their "homing" in adult individuals, is essential for sustained normal hemopoiesis. However, the specific molecules responsible for homing and for the continuing interaction of hemopoietic cells with the bone marrow stromal cells in vivo, or those involved in progenitor/stem cell trafficking through the bloodstream, have not been defined. A large repertoire of adhesion receptors, especially of the integrin family, appear to play a prominent role in promoting adhesion of hemopoietic stem cells to cultured marrow stromal cells in vitro. To test the functional role of cytoadhesion molecules in vivo, we treated primates systemically with either anti-alpha 4- or anti-beta 2-integrin antibodies, whose antigens are found in the majority of hemopoietic progenitors and in many differentiated cells. We found that anti-alpha 4 (anti-VLA4, anti-CD49d) but not anti-beta 2 (anti-CD18) treatment selectively mobilized progenitors into the bloodstream (up to 200-fold). Peripheralization involved erythroid, myeloid, and mixed progenitors; was detectable 24 hr after a single anti-VLA4 injection; and lasted beyond the days of treatment. Anti-VLA4 treatment additively augmented peripheralization of progenitors in animals with a preceding course of granulocyte-colony-stimulating factor. These data provide insight on the involvement of VLA4 antigens in the in vivo trafficking of progenitors and are of relevance to collection of peripheral blood stem cells for transplantation.
The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment.
Authors: Yang Y , Janich S , Cohn J A , Wilson J M
Published: May 2006

The most common cause of cystic fibrosis is deletion of Phe-508 (delta F508) from the cystic fibrosis transmembrane conductance regulator (CFTR). Previous studies have suggested that delta F508 CFTR is an unstable protein that retains a pattern of glycosylation specific to the endoplasmic reticulum. This report examines the mechanism responsible for the mislocalization of delta F508 CFTR in a human cystic fibrosis epithelial cell line overexpressing recombinant CFTR by virtue of adenovirus-mediated gene transfer. Immunoelectron microscopy confirmed that wild-type CFTR is delivered to the plasma membrane of these cells and that delta F508 CFTR is retained in the endoplasmic reticulum. Pulse-chase studies showed that newly synthesized CFTR complexes with the chaperone hsp70. The wild-type protein dissociates from hsp70 before its transport to the Golgi, and the protein is subsequently degraded in lysosomes. By contrast, the complex formed between delta F508 CFTR and hsp70 is retained in the endoplasmic reticulum and delta F508 CFTR is rapidly degraded in a pre-Golgi nonlysosomal compartment. Thus, hsp70 discriminates between the normal form of CFTR and the form of the protein that most commonly causes cystic fibrosis (delta F508). These findings clarify the mechanism by which mutation causing delta F508 affects the intracellular trafficking of CFTR and suggest another function for hsp70 in ensuring quality control during the biosynthesis of plasma-membrane proteins.
Daidzin and daidzein suppress free-choice ethanol intake by Syrian golden hamsters.
Authors: Keung W M , Vallee B L
Published: May 2006

Syrian Golden hamsters prefer and consume large and remarkably constant amounts of ethanol in a simple two-bottle free-choice regimen. Ethanol intake is significantly suppressed by zimelidine, bromocriptine, buspirone, and lithium carbonate, pharmacological agents that have been shown to be beneficial in controlling ethanol intake in alcohol-dependent humans. These results suggest that this ethanol-drinking animal model has high "predictive validity" and can be used effectively in the search for and identification of new agents for the treatment of alcohol abuse. The model has enabled us to confirm the putative antidipsotropic effect of Radix puerariae (RP), an herb long used in traditional Chinese medicine for the treatment of patients who abuse alcohol. A crude extract of RP at a dose of 1.5 g.kg-1 x day-1 significantly suppresses (> 50%) the free-choice ethanol intake of Golden hamsters. Moreover, two major constituents of RP, daidzein (4',7-dihydroxyisoflavone) and daidzin (the 7-glucoside of daidzein), were also shown to suppress free-choice ethanol intake. Daidzin and daidzein, at doses of 150 and 230 mg.kg-1 x day-1, respectively, suppress ethanol intake by > 50%. RP, daidzein, and daidzin treatment do not significantly affect the body weight and water or food intake of the hamsters. These findings identify a class of compounds that offer promise as safe and effective therapeutic agents for alcohol abuse.
The Src homology 2 domain of the protein-tyrosine kinase p56lck mediates both intermolecular and intramolecular interactions.
Authors: Amrein K E , Panholzer B , Flint N A , Bannwarth W , Burn P
Published: May 2006

A key event in signaling by many cell surface receptors is the activation of Src-like protein-tyrosine kinases and the assembly of protein complexes at the plasma membrane mediated by Src homology 2 and 3 (SH2 and SH3) domains. p56lck is a Src-related protein-tyrosine kinase which has SH2 and SH3 domains and is involved in T-cell signaling and oncogenic transformation. Here we demonstrate that purified recombinant SH2 and HSH3/SH2 domains of p56lck can mediate intermolecular interactions with a number of tyrosine-phosphorylated proteins present in lysates of NIH 3T3 cells transformed by a constitutively activated form of p56lck (p56lckF505). Two of the interacting tyrosine-phosphorylated proteins were identified as the p85 subunit of phosphatidylinositol 3-kinase and the GTPase-activating protein of p21ras. Using a synthetic phosphopeptide corresponding to the tyrosine-phosphorylated carboxyl terminus of p56lck (amino acids 494-509), purified recombinant Lck SH2 domain, and differentially phosphorylated forms of p56lck we provide evidence that the SH2 domain of p56lck can also mediate intramolecular interactions with the phosphorylated carboxyl terminus. Together these results suggest that the SH2 domain of p56lck has a dual function: (i) it can mediate intermolecular interactions with cellular proteins phosphorylated on tyrosine and thus might be involved in building up signaling complexes at the plasma membrane and (ii) it can bind to the tyrosine-phosphorylated carboxyl terminus of p56lck in an intramolecular fashion and thereby might be involved in the regulation of its intrinsic protein-tyrosine kinase activity. Phosphorylation/dephosphorylation of the regulatory tyrosine residue 505 might serve as a switch between these two functions.
26-10 Fab-digoxin complex: affinity and specificity due to surface complementarity.
Authors: Jeffrey P D , Strong R K , Sieker L C , Chang C Y , Campbell R L , Petsko G A , Haber E , Margolies M N , Sheriff S
Published: May 2006

We have determined the three-dimensional structures of the antigen-binding fragment of the anti-digoxin monoclonal antibody 26-10 in the uncomplexed state at 2.7 A resolution and as a complex with digoxin at 2.5 A resolution. Neither the antibody nor digoxin undergoes any significant conformational changes upon forming the complex. Digoxin interacts primarily with the antibody heavy chain and is oriented such that the carbohydrate groups are exposed to solvent and the lactone ring is buried in a deep pocket at the bottom of the combining site. Despite extensive interactions between antibody and antigen, no hydrogen bonds or salt links are formed between 26-10 and digoxin. Thus the 26-10-digoxin complex is unique among the known three-dimensional structures of antibody-antigen complexes in that specificity and high affinity arise primarily from shape complementarity.
Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli.
Authors: Altman J D , Reay P A , Davis M M
Published: May 2006

Class II major histocompatibility complex molecules play a major role in the immune response by binding peptide fragments of exogenous antigens and displaying them on the surfaces of antigen-presenting cells, where they can be recognized by T cells. To facilitate structural and functional studies of these molecules, we have produced truncated alpha and beta chains of the murine class II molecule I-Ek in Escherichia coli (Ec-I-Ek) and have developed conditions to fold them in the presence of specific peptides with yields of complex approaching 2%. Reconstitution is specific since only unlabeled peptide known to bind I-Ek compete with biotinylated peptide, as assessed by ELISA. Complexes of the refolded heterodimer (Ec-I-Ek) with either of two different peptide antigens remain associated during nonreducing SDS/PAGE. Immobilized Ec-I-Ek-peptide complexes stimulate lymphokine production by three T-cell clones in an antigen-specific manner with a dose-response relation comparable to previously described soluble I-Ek molecules produced in CHO cells. These results demonstrate that folding of Ek alpha and Ek beta polypeptides does not require any other protein to produce the biologically relevant conformation and that carbohydrate modification of this class II molecule is not necessary for alpha beta T-cell recognition.
Cloning of cDNA for the alpha subunit of mouse insulin-like growth factor I receptor and the role of the receptor in metanephric development.
Authors: Wada J , Liu Z Z , Alvares K , Kumar A , Wallner E , Makino H , Kanwar Y S
Published: May 2006

Various growth factors influence mammalian development by binding to specific cell surface receptors. These interactions are followed by a series of intracellular transductional events leading to a wide variety of biological effects. To establish the role of insulin-like growth factor I receptor (IGF-IR) in renal development, cDNA for the alpha subunit of the mouse IGF-IR was isolated, characterized, and used in expression studies and antisense experiments in a metanephric organ culture system. A 989-bp insert, encoding the signal peptide and 299 amino acids, isolated from a newborn mouse kidney cDNA library had 99% and 91% homology with the nucleotide sequences encoding the rat and the human IGF-IR, respectively. An approximately 11-kb message was readily detected by Northern blot analysis of RNA from the developing kidney at day 13 of gestation, and it declined during the subsequent embryonal and neonatal periods. In situ hybridization revealed high levels of message over the ureteric bud and its branches. A lower level of message was seen in the neonatal kidney, confined mainly to the tubules. Antisense oligodeoxynucleotide-treated metanephric kidneys were reduced in size and had a decreased population of nephrons with marked disorganization of ureteric bud branches. Immunofluorescence studies indicated an arrest of IGF-IR translation after antisense exposure. Immunoprecipitation studies showed a marked decrease in the biosynthesis of various extracellular matrix proteins that serve as regulators of morphogenesis. These studies suggest that the nucleotide sequence encoding the alpha subunit of mouse IGF-IR is highly conserved and that the receptor might play an essential role in the organogenesis of the kidney.
Escherichia coli ribosomal protein L7/L12 dimers remain fully active after interchain crosslinking of the C-terminal domains in two orientations.
Authors: Oleinikov A V , Jokhadze G G , Traut R R
Published: May 2006

Cysteine site-directed mutagenesis was used to create variants of Escherichia coli ribosomal protein L7/L12 that have single cysteine substitutions, at residues 63 or 89, located in different exposed loops in the structure of the globular C-terminal domain indicated by the crystallographic structure. That structure shows a possible dimer interaction in which the two sites of cysteine substitution appear to be too distant for disulfide bond formation. After mild oxidation in solution both of the overexpressed purified cysteine-substituted proteins formed interchain disulfide crosslinked dimers in high yield. Both crosslinked dimers were fully active in restoring activity in poly(U)-directed polyphenylalanine synthesis to ribosomal core particles depleted of wild-type L7/L12. These results show that the two C-terminal domains have independent mobility. The activity of dimeric L7/L12 does not require the independent movement of the two globular C-terminal domains in an L7/L12 dimer; moreover, it appears independent of their mutual orientation when joined by crosslinking at the two loops. A third variant with a cysteine substitution at residue 33 near the junction between the alpha-helical N-terminal domain and the flexible hinge was prepared and tested. This protein was active in the protein synthesis assay in the reduced state. Oxidation produced the interchain crosslinked dimer in high yield, but this crosslinked dimer was inactive in polyphenylalanine synthesis. The inactivation was due to the inability of the Cys33-Cys33 oxidized dimer to bind to the core particle.
Multiple oligomeric states regulate the DNA binding of helix-loop-helix peptides.
Authors: Fairman R , Beran-Steed R K , Anthony-Cahill S J , Lear J D , Stafford W F , DeGrado W F , Benfield P A , Brenner S L
Published: May 2006

To study the protein-protein interactions that allow Id, a negative regulator of cell differentiation, to inhibit the DNA-binding activities of MyoD and E47, we have synthesized peptides corresponding to the helix-loop-helix domains of MyoD, E47, and Id. We show that Id preferentially inhibits the sequence-specific DNA-binding activity of MyoD, a muscle-specific protein, as compared to E47, a more ubiquitous protein. The Id helix-loop-helix domain itself forms stable tetramers, and its inhibitory activity arises from the formation of a heterotetrameric structure with MyoD. The formation of this higher order complex provides a general mechanism by which inhibitory proteins can generate sufficient interaction free energy to overcome the large DNA-binding free energy of dimeric DNA-binding proteins.
Cloning of the human homolog of the CDC34 cell cycle gene by complementation in yeast.
Authors: Plon S E , Leppig K A , Do H N , Groudine M
Published: May 2006

In a screen designed to isolate human cDNAs that complement a yeast G2 phase checkpoint mutation (mec1), we isolated a cDNA homologous to the Saccharomyces cerevisiae CDC34 gene. The human CDC34 cDNA can functionally substitute for the yeast CDC34 gene and represents a mammalian homolog of the group of yeast genes required for the late G1-->S phase transition. The human CDC34 gene is expressed in multiple cell lines as a unique species and Southern blot analysis reveals evidence for a single gene that is highly conserved in higher eukaryotes. The human gene is located on the far telomeric region of 19p13.3 in a location that defines a region of homology between human chromosome 19p and mouse chromosome 11.
Electroporation of mammalian skin: a mechanism to enhance transdermal drug delivery.
Authors: Prausnitz M R , Bose V G , Langer R , Weaver J C
Published: May 2006

Mammalian skin owes its remarkable barrier function to its outermost and dead layer, the stratum corneum. Transdermal transport through this region occurs predominantly through intercellular lipids, organized largely in bilayers. Electroporation is the creation of aqueous pores in lipid bilayers by the application of a short (microseconds to milliseconds) electric pulse. Our measurements suggest that electroporation occurs in the intercellular lipid bilayers of the stratum corneum by a mechanism involving transient structural changes. Flux increases up to 4 orders of magnitude were observed with human skin in vitro for three polar molecules having charges between -1 and -4 and molecular weights up to slightly more than 1000. Similar flux increases were observed in vivo with animal skin. These results may have significance for drug delivery and other medical applications.
Genetic analysis of homomeric interactions of human immunodeficiency virus type 1 integrase using the yeast two-hybrid system.
Authors: Kalpana G V , Goff S P
Published: May 2006

The retroviral integrase protein (IN) is responsible for catalyzing a concerted integration reaction in which the two termini of linear viral DNA are joined to host DNA. To probe the potential for IN to form protein multimers, we used the yeast two-hybrid system. The coexpression of a GAL4 DNA binding domain-IN fusion and a GAL4 activation domain-IN fusion together resulted in the successful activation of a GAL4-responsive LacZ reporter gene. The system was used to examine a variety of IN deletion mutants. The results suggest that the central region of the protein is necessary for multimerization and that the N-terminal zinc finger region is not important.
Multiple loci for synapse protein SNAP-25 in the tetraploid goldfish.
Authors: Risinger C , Larhammar D
Published: May 2006

The common goldfish Carassius auratus is tetraploid and has 100 chromosomes. We describe here goldfish cDNA clones for SNAP-25, a 200-amino-acid synaptosome-associated protein that has remained highly conserved during evolution. SNAP-25 occurs as a single-copy gene in mouse, chicken, and Drosophila melanogaster. Sequences of six distinct goldfish cDNA clones and Southern hybridizations show that the goldfish has three, or possibly four, SNAP-25 loci rather than two as expected. A gene duplication early in actinopterygian fish evolution gave rise to the loci SnapA and SnapB. The proteins SNAP-A and SNAP-B are 94% and 91% identical to the mouse protein but are only 91% identical to each other. SNAP-B has a larger number of unique amino acid replacements than SNAP-A and also has more dramatic replacements. The tetraploidization resulted in two SnapB loci whose divergence from each other is consistent with a tetraploidization event 15-20 million years ago. The presence of duplicate SnapA loci has not yet been possible to confirm, possibly because they are still very similar to each other. Two of the SnapA cDNA clones and one SnapB cDNA clone have frameshift mutations. As these aberrant alleles otherwise display high sequence identity to the functional alleles, they probably became nonfunctional recently. The findings of allelic variability and aberrant alleles emphasize the importance of characterizing multiple DNA clones in tetraploid species.
Heterotypic and homotypic associations between ezrin and moesin, two putative membrane-cytoskeletal linking proteins.
Authors: Gary R , Bretscher A
Published: May 2006

Ezrin and moesin are components of actin-rich cell surface structures that are thought to function as membrane-cytoskeletal linking proteins. Here we show that a stable complex of ezrin and moesin can be isolated from cultured cells by immunoprecipitation with specific antibodies. The capacity of these two proteins to interact directly was confirmed with a blot-overlay procedure in which biotin-tagged proteins in solution were incubated with immobilized binding partners. In addition to the heterotypic association of ezrin and moesin, homotypic binding of ezrin to ezrin and of moesin to moesin was also demonstrated in vitro. These results suggest mechanisms by which ezrin and moesin might participate in dynamic aspects of cortical cytoskeletal structure.