bluuebunny/crossref_metadata_2025 huggingface dataset:
Human skin fibroblasts in vitro differentiate along a terminal cell lineage.
Authors: Bayreuther K , Rodemann H P , Hommel R , Dittmann K , Albiez M , Francz P I
Published: May 2006

Secondary mitotic human skin fibroblast populations in vitro underwent 53 +/- 6 cumulative population doublings (CPD) in 302 +/- 27 days. When the growth capacity of the mitotic fibroblasts is exhausted, and if appropriate methods are applied, the fibroblasts differentiate spontaneously into postmitotic fibroblast populations, which were kept in stationary culture for up to 305 +/- 41 additional days. Mitotic and postmitotic fibroblast populations are heterogeneous populations with reproducible changes in the proportions of mitotic fibroblasts F I, F II, and F III, and postmitotic fibroblasts F IV, F V, F VI, and F VII. This process makes it evident that the fibroblasts differentiate spontaneously along a seven-stage terminal cell lineage F I-F II-F III-F IV-F V-F VI-F VII. Shifts in the frequencies of the mitotic and postmitotic fibroblasts in mass populations are accompanied by alterations in the [35S]methionine polypeptide pattern of the developing mass populations. The [35S]methionine polypeptide patterns of homogeneous subpopulations of F I, F II, F III, F IV, F V, and F VI isolated from heterogeneous mass populations reveal that the six fibroblast morphotypes studied express their cell-type-specific [35S]methionine polypeptide pattern in the heterogeneous mass populations.
Quantitative analysis of RNA produced by slow and fast alleles of Adh in Drosophila melanogaster.
Authors: Laurie C C , Stam L F
Published: May 2006

The alcohol dehydrogenase (ADH) locus (Adh) of Drosophila melanogaster in polymorphic on a world-wide basis for two allozymes, Fast and Slow. This study was undertaken to determine whether the well-established difference in ADH protein concentration between the allozymes is due to a difference in mRNA levels. RNA gel blot hybridization and an RNase protection assay were used to quantify ADH mRNA levels. Each method used an Adh null mutant as an internal standard. Several Slow and Fast allele pairs of different geographic origins were analyzed. The results provide strong evidence that the ADH protein concentration difference is not accounted for by RNA level.
The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.
Authors: Rudd C E , Trevillyan J M , Dasgupta J D , Wong L L , Schlossman S F
Published: May 2006

The CD4 (T4) antigen is a cell-surface glycoprotein that is expressed predominantly on the surface of helper T cells and has been implicated in the regulation of T-cell activation and in the associative recognition of class II antigens of the major histocompatibility complex. In addition, the CD4 antigen appears to serve as a receptor for the human immunodeficiency virus (HIV). An important question has been whether the CD4 receptor is linked to an intracellular mediator that could regulate the activation of the CD4+ subset. In this paper, we provide preliminary evidence that the CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (PTK) of 55-60 kDa, which is expressed specifically in T cells. The PTK is the human analogue of the murine pp56LSTRA (pp56lck) and has significant homology with c-src, c-yes, and other members of the src family. The identification of the PTK associated with CD4 receptor was made by use of an antiserum to a synthetic peptide that was deduced from the DNA sequence of PTK. Two-dimensional nonequilibrium pH gradient gel electrophoresis/NaDodSO4/PAGE revealed the kinase to focus as a heterogeneous collection of spots in the pH range of 4.0-5.0. Furthermore, in vitro phosphorylation revealed the phosphorylation of two additional polypeptides at 40 and 80 kDa, in addition to the autophosphorylation of the PTK at 55-60 kDa. The potential importance of the association between the CD4 receptor and the PTK of T cells is discussed in relation to T-cell activation and HIV infectivity.
Activation of type B gamma-aminobutyric acid receptors in the intact mammalian spinal cord mimics the effects of reduced presynaptic Ca2+ influx.
Authors: Lev-Tov A , Meyers D E , Burke R E
Published: May 2006

Intracellular recordings from mammalian spinal motoneurons in vivo show that the type B gamma-aminobutyric acid receptor agonist, L-(-)-baclofen, when administered systemically to pentobarbital-anesthetized or decerebrate unanesthetized cats decreases the amplitude of monosynaptic group Ia excitatory postsynaptic potentials (EPSPs), markedly increases tetanic and posttetanic potentiation, and reduces or abolishes synaptic depression during high-frequency synaptic activation and in the posttetanic period. These changes occur without detectable alteration in motoneuron input resistance, EPSP shape, or the invasion of action potentials into the intraspinal group Ia terminal arborizations. The baclofen-induced effects are qualitatively similar to those observed in more accessible synaptic systems when presynaptic Ca2+ influx and, concomitantly, transmitter release are reduced. Based on these and other recent findings regarding the mechanism of action of baclofen and the distribution of its receptors in the spinal cord, we suggest that L-(-)-baclofen modifies frequency modulation of Ia synaptic transmission by reducing presynaptic Ca2+ influx and the concomitant level of transmitter release from Ia afferent terminals. The drug appears to be a useful tool in studies of the ionic mechanisms that control the release of transmitter and its frequency modulation at inaccessible mammalian synapses.
Homotopy invariance of η-invariants
Authors: Weinberger Shmuel
Published: May 2006

Intersection homology and results related to the higher signature problem are applied to show that certain combinations of η-invariants of the signature operator are homotopy invariant in various circumstances.
In vivo "photofootprint" changes at sequences between the yeast GAL1 upstream activating sequence and "TATA" element require activated GAL4 protein but not a functional TATA element.
Authors: Selleck S B , Majors J
Published: May 2006

Transcription of the yeast GAL1 and GAL10 genes is induced by growth on galactose. Using the technique of photofootprinting in vivo, we previously documented equivalent transcription-dependent footprints within the putative "TATA" elements of both genes. To explore the functional significance of these observations, we created a 3-base-pair substitution mutation within the GAL1 promoter TATA element, which disrupted the ATATAA consensus sequence but left intact the photomodification targets. The mutation reduced galactose-induced RNA levels by a factor of 100. The mutant promoter no longer displayed the characteristic TATA sequence footprint, supporting the hypothesis that transcription activation involves the binding of a TATA box factor. We also observed a collection of transcription-correlated alterations in the modification pattern at sites between the UASG and the GAL1 TATA element, within sequences that are not required for inducible transcription. These patterns, characteristic of the induced wild-type GAL1 gene, were still galactose inducible with the TATA mutant GAl1 promoter, despite the low level of transcription from this promoter. We conclude that the GAL4-dependent protein/DNA structure responsible for the altered pattern within nonessential sequences is therefore not strictly coupled to an active TATA element or to high levels of expression. Nonetheless, the patterns probably reflect a stable protein-dependent structure that accompanies assembly of the transcription initiation complex.
Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label.
Authors: Miller W T , Kaiser E T
Published: May 2006

A peptide-based photoaffinity label for the catalytic subunit of the cAMP-dependent protein kinase was prepared from the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)]. By using solid-phase peptide synthesis methodology, DL-Phe(pBz) was incorporated into the cAMP-dependent protein kinase substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in place of the phosphorylatable serine. The diastereomeric peptides were separated by reverse-phase HPLC. The peptide substrate analog containing L-Phe(pBz) had a Ki of approximately 110 microM at pH 7.5. When photolyzed at 350 nm in the presence of the enzyme, this peptide caused time- and concentration-dependent inactivation. Radioactive acetylated L-Phe(pBz) peptide was used to establish the binding stoichiometry of peptide to enzyme; these results, together with protection experiments, showed the photoaffinity labeling to be specific (approximately 1:1). To identify the residues that were modified on the catalytic subunit, the photoinactivated enzyme was cleaved with CNBr and V8 protease (Staphylococcus aureus). The resulting peptide fragments were purified by HPLC and were sequenced; these experiments identified the modified residues as Gly-125 and Met-127. This region of the cAMP-dependent protein kinase catalytic subunit contains many residues that are conserved in serine- and tyrosine-protein kinases.
Activation and expansion of hapten- and protein-specific T helper cells from nonsensitized mice.
Authors: Hauser C , Katz S I
Published: May 2006

Hapten- and protein-antigen-specific T helper cells are usually expanded in vitro from lymphocytes obtained from sensitized animals. In this paper we report on the primary activation and proliferation in vitro of T helper cells from nonsensitized animals by using syngeneic cultured epidermal Langerhans cells as a source of potent antigen-presenting cells. The primary in vitro proliferation was blocked with monoclonal antibodies to Ia molecules, to lymphocyte function-associated antigen 1 (LFA-1), and to L3T4. T helper cell populations sensitized in vitro to haptens and protein antigens showed hapten- and antigen-specific proliferation when restimulated in vitro with spleen cells. Besides its experimental usefulness, in vitro generation of syngeneic specific T helper cells may afford possibilities for adoptive immunotherapy.
Differential loss of striatal projection neurons in Huntington disease.
Authors: Reiner A , Albin R L , Anderson K D , D'Amato C J , Penney J B , Young A B
Published: May 2006

Huntington disease (HD) is characterized by the loss of striatal projection neurons, which constitute the vast majority of striatal neurons. To determine whether there is differential loss among different populations of striatal projection neurons, the integrity of the axon terminal plexuses arising from the different populations of substance P-containing and enkephalin-containing striatal projection neurons was studied in striatal target areas by immunohistochemistry. Analysis of 17 HD specimens indicated that in early and middle stages of HD, enkephalin-containing neurons projecting to the external segment of the globus pallidus were much more affected than substance P-containing neurons projecting to the internal pallidal segment. Furthermore, substance P-containing neurons projecting to the substantia nigra pars reticulata were more affected than those projecting to the substantia nigra pars compacta. At the most advanced stages of the disease, projections to all striatal target areas were depleted, with the exception of some apparent sparing of the striatal projection to the substantia nigra pars compacta. These findings may explain some of the clinical manifestations and pharmacology of HD. They also may aid in identifying the neural defect underlying HD and provide additional data with which to evaluate current models of HD pathogenesis.
Cloning, nucleotide sequence, and potential regulatory elements of the glutamine synthetase gene from murine 3T3-L1 adipocytes.
Authors: Bhandari B , Beckwith K D , Miller R E
Published: May 2006

Glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] specific activity, cellular content, mRNA abundance, and gene transcription rate increase by greater than 100-fold during adipocyte differentiation of 3T3-L1 cells. In 3T3-L1 adipocytes dexamethasone increases, whereas insulin as well as N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate decrease, glutamine synthetase gene expression. We analyzed the nucleotide sequence of a 1.9-kilobase Sal I-EcoRI restriction fragment from a 3T3-L1 glutamine synthetase genomic clone. This genomic fragment is composed of 1851 base pairs (bp) and includes the first exon and 1029 bp of the 5' flanking sequence. The 600 bp at the 3' end of the 1.9-kb Sal I-EcoRI restriction fragment constitute an open reading frame. We identified the transcription start site at a location 222 bp upstream of the glutamine synthetase coding sequences. The 5' flanking region of the gene encompasses several potential regulatory elements including TATA and CAAT sequences and a 40-bp poly(dT-dG).poly(dC-dA) putative enhancer element. Potential hormone and fat-specific regulatory elements are also located upstream of the transcription start site; they include glucocorticoid and cAMP response elements and fat-specific elements. These potential regulatory elements could account for the differentiation-associated changes and hormone-mediated changes seen in glutamine synthetase gene transcription and mRNA abundance.
Structure of the carboxyl terminus of the RAS gene-encoded P21 proteins.
Authors: Brandt-Rauf P W , Carty R P , Chen J , Avitable M , Lubowsky J , Pincus M R
Published: May 2006

The three-dimensional structures of the carboxyl-terminal regions of the P21 protein products of the human Harvey (Ha), Kirsten (KiA and KiB), and neuroblastoma (N) RAS oncogenes and various mutants have been determined by using conformational energy analysis. The carboxyl-terminal region of P21 has been strongly implicated in the binding of the protein to the inner surface of the plasma membrane without which the protein is inactive. The only invariant residue in this region is Cys-186, which is necessary for the post-translational addition of palmitic acid. The surrounding sequences of the active native proteins differ considerably. Nevertheless, certain amino acid substitutions in this region are known to eliminate membrane binding and protein activity, suggesting that there is a conserved common structural feature in this region in the native proteins that is disrupted in the mutant proteins. Conformational energy analysis shows that the four native P21 proteins have a common structure in the form of an alpha-helix for the terminal pentapeptide. A mutant, pBW277, that fails to bind to the membrane and is inactive cannot adopt an alpha-helical structure in this region because of a proline at position 188. Another mutant, pBW766, that retains membrane binding and activity, on the other hand, retains the preference for an alpha-helical conformation in the terminal pentapeptide. These findings suggest that, despite various amino acid sequences in this region, the carboxyl-terminal pentapeptides of the P21 proteins form a distinctive structural domain that must have an alpha-helical structure for membrane binding and intracellular activity.
Selective stabilization of microtubules oriented toward the direction of cell migration.
Authors: Gundersen G G , Bulinski J C
Published: May 2006

A small subset of the microtubule (MT) array in many cultured cells does not exhibit the rapid turnover (t 1/2 approximately equal to 10 min) shown by most cellular MTs. The function of the stable class of MTs is unknown and has been confounded by the apparent lack of organization of stable MTs within cells. Using an antibody against detyrosinated tubulin, a post-translationally modified form of tubulin that accumulates in stable MTs, we localized the stable MTs in mouse 3T3 cells induced to initiate directional migration by experimental wounding of confluent monolayers. Immediately after monolayer wounding, the distribution of stable MTs in cells at the wound edge resembled that in cells in the monolayer interior; most cells either contained randomly distributed stable MTs or lacked them entirely. However, by 20 min after wounding, cells at the wound margin began to generate an asymmetric MT array, with virtually all stable MTs oriented toward the cell edge in contact with the wound. Two hours after monolayer wounding, greater than or equal to 80% of cells at the wound margin had generated this polarized array of stable MTs, and the array was maintained for at least 12 hr. MTs in the polarized array showed enhanced resistance to depolymerization by nocodazole, thus providing an independent test of their stability. Formation of the polar array of stable MTs appeared to precede onset of cell migration and closely paralleled reorientation of the MT-organizing center. These results show that cultured cells can remodel their MT array rapidly in response to an extracellular signal and suggest that selective stabilization of MTs is an early event in the generation of cellular asymmetry.
Specific binding of murine leukemia inhibitory factor to normal and leukemic monocytic cells.
Authors: Hilton D J , Nicola N A , Metcalf D
Published: May 2006

Leukemia inhibitory factor (LIF), a glycoprotein capable of suppressing the clonogenicity and inducing the differentiation of the murine myeloid leukemia cell line M1, was radioiodinated to a high specific radioactivity with retention of full biological activity. Binding of 125I-labeled LIF to M1 cells reached a steady state at 37 degrees C after approximately equal to 40 min and was in competition with unlabeled LIF but not granulocyte colony-stimulating factor or a range of other cytokines or differentiation-inducing agents. Specific binding was demonstrable to cells from a range of murine hemopoietic tissues including the bone marrow, the spleen, and the peritoneal cavity. Autoradiography revealed macrophages, monocytes, and their precursors to be the major cell types responsible for 125I-labeled LIF binding within these tissues. Receptors on M1 cells were of high affinity (apparent Kd, 100-200 pM) and few in number (300-500 per cell).
Expression of the human beta-globin gene following retroviral-mediated transfer into multipotential hematopoietic progenitors of mice.
Authors: Karlsson S , Bodine D M , Perry L , Papayannopoulou T , Nienhuis A W
Published: May 2006

Efficient transfer of the beta-globin gene into primitive hematopoietic progenitors was achieved with consistent and significant expression in the progeny of those cells. Retroviral vectors containing the intact genomic human beta-globin gene and the neomycin (G418)-resistance (neoR) gene were constructed. These gave titers of 10(6) or more neoR colony-forming units/ml when packaged in psi 2 cells. Mouse bone marrow cells were infected by coculture with producer cells and injected into lethally irradiated animals. Several parameters were varied to enhance infection frequency of colony-forming units, spleen (CFU-S); overall 41% of 116 foci studied contained an intact proviral genome. The human beta-globin gene was expressed in 31 of 35 CFU-S-derived spleen colonies that contained the intact vector genome at levels ranging from 1% to 5% of that of the mouse beta-globin genes. Infected bone marrow cells were also injected into genetically anemic W/Wv recipients without prior irradiation. Human beta-globin chains were detected in circulating erythrocytes by immunofluorescent staining with a specific monoclonal antibody. All animals injected with donor cells that had been cultured in G418 (1 mg/ml) for 48 hr after retroviral infection had circulating erythrocytes containing human beta-globin chains between 3 and 8 weeks after transplantation.
Anti-idiotypic antibodies that react with microtubule-associated proteins are present in the sera of rabbits immunized with synthetic peptides from tubulin's regulatory domain.
Authors: Rivas C I , Vera J C , Maccioni R B
Published: May 2006

A fundamental question in microtubule research is how the interactions of tubulin subunits with microtubule-associated proteins (MAPs) are controlled. The answer should provide insight into the regulation of the cellular processes in which microtubules are implicated. Previous work demonstrated the interaction of MAPs with a 4-kDa C-terminal domain of tubulin alpha and beta subunits. Synthetic peptides from the variable region of the 4-kDa C-terminal moiety of tubulin subunits, alpha-(430-441) and beta-(422-434), bind to MAP-2 and to the MAP tau, and a preferential interaction of the beta peptide is observed. To define the regulatory significance of the substructure of the C-terminal tubulin domain, we produced rabbit antisera against these MAP-interacting peptides. We found that these antisera contained not only antibodies to the original synthetic peptides but also antibodies to MAPs. Here, we report that these antibodies, which react with MAP-1, MAP-2, and tau, appear to be a population of anti-idiotypic antibodies directed to the anti-peptide antibodies. They can inhibit MAP-induced tubulin assembly into microtubules in vitro, and the addition of MAPs overcomes the inhibition. The recognition by these anti-idiotypic antibodies of the tubulin-binding domain on MAPs provides unequivocal evidence that the tubulin region defined by the synthetic peptides is directly involved in the interaction with MAPs.
Atrial-specific granules in situ have high calcium content, are acidic, and maintain anion gradients.
Authors: Somlyo A V , Broderick R , Shuman H , Buhle E L , Somlyo A P
Published: May 2006

The composition and pH of atrial-specific granules of rat heart were determined by electron probe x-ray microanalysis and fluorescence microscopy, respectively. The high (75 mmol/kg of dry weight) calcium content and higher than cytoplasmic concentration of chloride in atrial-specific granules were visualized in high-resolution x-ray maps. The Cl- content of granules and cytoplasm decreased and a bromide gradient (granule greater than cytoplasm) was established during incubation in low-chloride, NaBr-containing solutions. Scanning confocal fluorescence light microscopy of live atria incubated with acridine orange demonstrated dye accumulation, indicative of low intragranular pH. We conclude that the granules represent a hitherto unrecognized intracellular store of cardiac calcium and can develop and maintain an anion gradient, presumably through cotransport by means of a proton-pumping ATPase.
Integration host factor interacts with the DNA replication enhancer of filamentous phage f1.
Authors: Greenstein D , Zinder N D , Horiuchi K
Published: May 2006

We present data which show that the Escherichia coli integration host factor (IHF) is an activator of phage f1 DNA replication. Phage f1 poorly infects bacterial strains lacking IHF because IHF is required for efficient expression of F-pili, the receptor for f1 phage. However, when F- strains are transfected with f1 DNA the phage replicates in IHF mutants (himA, himD, or himA himD) at a rate of only 3% of that in wild-type bacteria. A plasmid dependent on the f1 replicon fails to transform IHF mutants. By gel retardation analysis, we show that IHF specifically binds to the origin of replication. DNase I "footprinting" experiments demonstrate that IHF binds to multiple sites within the replication enhancer sequence, a cis-acting, A + T-rich sequence that potentiates f1 DNA replication. Moreover, the effect of IHF mutation on f1 growth is suppressed by initiator protein (f1 gene II) mutations that restore efficient replication from origins that lack a functional replication enhancer sequence. This genetic evidence supports the conclusion that the replication enhancer sequence is the site of action of IHF.
Mutational analysis of the chicken beta-globin enhancer reveals two positive-acting domains.
Authors: Reitman M , Felsenfeld G
Published: May 2006

We report a mutational analysis of the chicken beta-globin enhancer, using transient expression in primary embryonic erythrocytes. A series of 27 scanning mutants differing only by consecutive 4-base substitutions was tested. Previous experiments, using protection from DNase I digestion, demonstrated four regions in the enhancer that bind factors. Mutations in two of the four regions (denoted I and III) have no effect on enhancer activity. The other two regions (II and IV) mediate all of the positive-acting activity. Region II appears to consist of two subregions, mutation in either of which leads to the same loss in activity as mutation of both. Like region II, mutation of either half of region IV results in reduced enhancer activity. However, in contrast to region II, mutation of both halves of region IV results in more loss of enhancer activity than either single mutation. These data suggest that the half-sites in region II must interact in order to cause enhancement, whereas the half-sites in region IV act independently. Triplications of regions II or IV are able to enhance to a similar degree as the parental enhancer, but single copies of regions II or IV enhance only weakly. Thus, as measured by transient expression in embryonic erythrocytes, the beta-globin enhancer may involve the action of as few as three DNA-binding proteins acting at two sites.
Molecular cloning and sequencing of DNA complementary to chicken liver fatty acid synthase mRNA.
Authors: Yuan Z Y , Liu W , Hammes G G
Published: May 2006

The cDNA corresponding to 4.18 kilobases (kb) of the mRNA of chicken liver fatty acid synthase has been cloned and sequenced. The cDNA corresponds to the 3' end of the mRNA and consists of a 1.87-kb noncoding tail and a 2.31-kb region encoding 769 amino acids of the C terminus of the enzyme. The thioesterase at the C terminus, preceded by the acyl carrier protein, can be identified from known amino acid sequences. However, the identity of the enzymes N terminal to the acyl carrier protein could not be ascertained. The partial amino acid sequence of the chicken liver fatty acid synthase shows greater than 70% similarity with the rat mammary gland enzyme.
Location of the genes controlling H-Y antigen expression and testis determination on the mouse Y chromosome.
Authors: McLaren A , Simpson E , Epplen J T , Studer R , Koopman P , Evans E P , Burgoyne P S
Published: May 2006

Sex-reversed XX male mice that carry the variant form of the testis-determining Sxr region, Sxr', do not express male-specific H-Y antigen. In a stock of mice segregating for Sxr', we detected an exceptional XX male that proved positive for H-Y antigen. DNA fingerprinting revealed that the banding pattern characteristic of Sxr' had been replaced by the pattern associated with the native testis-determining region of the normal Y chromosome of that stock, presumably by pairing and crossing-over between the two testis-determining regions of the father's Y Sxr' chromosome. Pairing between the two ends of such a chromosome in a loop-like configuration has been observed by electron microscopy. However, an anomalous crossing-over event of this kind would only give rise to the observed result if the native homologue of the Sxr region were situated on the very minute short arm of the Y chromosome. We therefore conclude that the two linked genes Tdy and Hya, controlling testis determination and H-Y antigen expression, respectively, are located on the short arm of the mouse Y chromosome.
Identification of the receptor for erythropoietin on human and murine erythroleukemia cells and modulation by phorbol ester and dimethyl sulfoxide.
Authors: Broudy V C , Lin N , Egrie J , de Haën C , Weiss T , Papayannopoulou T , Adamson J W
Published: May 2006

Erythropoietin, a glycoprotein that regulates erythropoiesis, initiates its biological effects by binding to a cell-surface receptor. Little is known about the structure of the erythropoietin receptor and the events that follow binding of erythropoietin to its receptor, in part because of the difficulty of obtaining sufficient quantities of cells that express the erythropoietin receptor. We used both iodinated and metabolically labeled erythropoietin to characterize the receptor on a variety of erythroleukemia cell lines not previously tested, and we have identified both human and murine cell lines that display large numbers of erythropoietin receptors. Both erythropoietin-responsive and -nonresponsive cell lines exhibit a single class of binding sites. The human erythroleukemia cell line OCIM1 exhibits approximately 3000 erythropoietin receptors per cell with a Kd of 280 pM. The erythropoietin-responsive Rauscher red 5-1.5 murine erythroleukemia cell line displays approximately 1700 receptors per cell with a Kd of 440 pM. The GM979 murine erythroleukemia cell line has approximately 1600 receptors per cell with a Kd of 660 pM. Induction of the erythroid phenotype by dimethyl sulfoxide or its suppression by phorbol 12-myristate 13-acetate was accompanied by an increase or decrease, respectively, in erythropoietin receptor number. Affinity crosslinking of labeled erythropoietin to the receptor identified two proteins corresponding to estimated molecular masses of 95 and 105 kDa. The OCIM1, Rauscher, and GM979 erythroleukemia cell lines provide a useful model for the study of postreceptor signaling events, as well as a convenient source for purification of the erythropoietin receptor.
7-Chlorokynurenic acid is a selective antagonist at the glycine modulatory site of the N-methyl-D-aspartate receptor complex.
Authors: Kemp J A , Foster A C , Leeson P D , Priestley T , Tridgett R , Iversen L L , Woodruff G N
Published: May 2006

Glycine markedly potentiates N-methyl-D-aspartate (N-Me-D-Asp) responses in mammalian neurons by an action at a modulatory site on the N-Me-D-Asp receptor-ionophore complex. Here we present evidence that 7-chlorokynurenic acid (7-Cl KYNA) inhibits N-Me-D-Asp responses by a selective antagonism of glycine at this modulatory site. In rat cortical slices 7-Cl KYNA (10-100 microM) noncompetitively inhibited N-Me-D-Asp responses, and this effect could be reversed by the addition of glycine (100 microM) or D-serine (100 microM). Radioligand binding experiments showed that 7-Cl KYNA had a much higher affinity for the strychnine-insensitive [3H]glycine binding site (IC50 = 0.56 microM) than for the N-Me-D-Asp (IC50 169 microM), quisqualate (IC50 = 153 microM), or kainate (IC50 greater than 1000 microM) recognition sites. In whole-cell patch-clamp recordings from rat cortical neurones in culture, the inhibitory effects of 7-Cl KYNA on N-Me-D-Asp-induced currents could not be overcome by increasing the N-Me-D-Asp concentration but could be reversed by increasing the glycine concentration. 7-Cl KYNA could completely abolish N-Me-D-Asp responses, including basal responses in the absence of added glycine, suggesting that it may possess negative modulatory effects at the glycine site. These findings indicate that the glycine modulatory site is functional in intact adult tissue and that 7-Cl KYNA should prove to be a selective tool for elucidating the involvement of this site in physiological and pathological events mediated by N-Me-D-Asp receptors.
RETRACTED: Photoreduction of carbon dioxide by aqueous ferrous ion: An alternative to the strongly reducing atmosphere for the chemical origin of life
Authors: Borowska Zofia , Mauzerall David
Published: May 2006

We have shown that ferrous ion at neutral pH photoreduces water to hydrogen with a high quantum yield on excitation with near-ultraviolet light. This simple system also efficiently reduces carbon dioxide (bicarbonate ions) to formaldehyde. Overall, these reactions offer a solution to a dilemma confronting the standard or Oparin-Urey model of the origin of life. If carbon dioxide was the main form of carbon on the primitive earth, the ferrous photoreaction would have provided the reduced carbon necessary to form amino acids and other biogenetic molecules. We believe this system may have been the progenitor to the biological photosynthetic systems.
Cytochrome c oxidase from Paracoccus denitrificans: both hemes are located in subunit I.
Authors: Müller M , Schläpfer B , Azzi A
Published: May 2006

The two-subunit cytochrome c oxidase from Paracoccus denitrificans has been sequentially digested with chymotrypsin and Staphylococcus aureus V8 protease. The smaller subunit of the enzyme (apparent Mr 32,000) was split into numerous peptides that were removed by anion-exchange HPLC. The larger subunit was only digested to a limited extent (from an apparent Mr 45,000 to Mr 43,000), and the spectral properties were preserved relative to the native enzyme (a reduced minus oxidized difference spectrum with maxima at 447 and 607 nm in the Soret and alpha region, respectively). As judged from CO-reduced spectra this proteolytically digested, one-fragment oxidase was found to contain an equal amount of cytochromes a and a3. The enzymatic activity with reduced cytochrome c as substrate in the presence of Triton X-100 proceeded with equal affinity (apparent Km = 0.5-1.0 microM) and with a Vmax of approximately 20% (40 s-1) of that found with the native enzyme (200 s-1). When the assay system was supplemented with soybean phospholipids, the Km became 2 microM for both enzymes and the Vmax became 730 and 170 s-1 for the native and the digested enzyme, respectively. Thus subunit I of P. denitrificans oxidase, and most probably of the other cytochrome c oxidases as well, contains both hemes and at least one Cu atom and has significant enzymatic activity.
Characterization of a gene from a tomato pathogen determining hypersensitive resistance in non-host species and genetic analysis of this resistance in bean
Authors: Whalen Maureen C. , Stall Robert E. , Staskawicz Brian J.
Published: May 2006

Xanthomonas campestris
pv.
vesicatoria
is the causal agent of leaf spot disease on pepper and tomato. On non-host plants, such as bean, soybean, cowpea, alfalfa, and cotton,
X. campestris
pv.
vesicatoria
is unable to cause disease, inducing instead a hypersensitive resistance response (HR). Since avirulence genes from
X. campestris
pv.
vesicatoria
specifically induce HR in several pepper cultivars, we investigated whether there were avirulence genes governing induction of resistance in non-host species. We report on the molecular cloning and characterization of a non-host avirulence gene from
X. campestris
pv.
vesicatoria
. A cosmid clone isolated from a library of DNA from
X. campestris
pv.
vesicatoria
tomato race 1 converted
X. campestris
pv.
phaseoli
to avirulence by inducing HR on the bean cultivar Sprite, but not on Bush Blue Lake. The HR-inducing activity was localized to a 2.1-kilobase
Pst
I fragment of DNA, designated
avrRxv
. In addition, we demonstrate that
avrRxv
inhibited disease production by several
X. campestris
pathovars on their normally susceptible hosts:
glycines
on soybean,
vignicola
on cowpea,
alfalfae
on alfalfa,
holcicola
on corn, and
malvacearum
on cotton. The HR resistance in bean induced by
avrRxv
segregated as a single incompletely dominant gene, designated
Rxv
. These results indicate that the avirulence gene
avrRxv
and the resistance gene
Rxv
partially control the outcome of the interaction between
X. campestris
pv.
vesicatoria
and the non-host bean.
Albumin interacts specifically with a 60-kDa microvascular endothelial glycoprotein.
Authors: Schnitzer J E , Carley W W , Palade G E
Published: May 2006

Confluent monolayers of microvascular endothelial cells, derived from the rat epididymal fat pad and grown in culture, were radioiodinated by using the lactoper-oxidase method. Their radioiodinated surface polypeptides were detected by NaDodSO4/PAGE (followed by autoradiography) and were characterized by both lectin affinity chromatography and protease digestion to identify the proteins involved in albumin binding. All detected polypeptides were sensitive to Pronase digestion, whereas several polypeptides were resistant to trypsin. Pronase treatment of the cell monolayer significantly reduced the specific binding of radioiodinated rat serum albumin, but trypsin digestion did not. Limax flavus, Ricinus communis, and Triticum vulgaris agglutinins competed significantly with radioiodinated rat serum albumin binding, whereas other lectins did not. A single 60-kDa glyco-protein was precipitated in common by these three lectins and was trypsin-resistant and Pronase-sensitive. Rat serum albumin affinity chromatography columns weakly but specifically bound a 60-kDa polypeptide from cell lysates derived from radioiodinated cell monolayers. These findings indicate that the 60-kDa glycoprotein is directly involved in a specific interaction of albumin with the cultured microvascular endothelial cells used in these experiments.
Gene encoding capsid protein VP1 of foot-and-mouth disease virus: a quasispecies model of molecular evolution.
Authors: Dopazo J , Sobrino F , Palma E L , Domingo E , Moya A
Published: May 2006

A phylogenetic tree relating the VP1 gene of 15 isolates of foot-and-mouth disease virus (FMDV) of serotypes A, C, and O has been constructed. The most parsimonious tree shows that FMDV subtypes and isolates within subtypes constitute sets of related, nonidentical genomes, in agreement with a quasispecies mode of evolution of this virus. The average number of nucleotide replacements per site for all possible pairs of VP1 coding segments is higher among representatives of serotype A than serotype C or O. In comparing amino acid sequences, the values of dispersion index (variance/mean value) are greater than 1, with the highest values scored when all sequences are considered. This indicates an accumulation of mutations at a limited number of residues, suggesting that distributions of sequences fluctuate around points of high stability. Evolution of FMDV follows a path very distant from that of a star phylogeny, and it has not been possible to derive conclusions on constancy of evolutionary rates with the test applied to the analysis. FMDVs, as other RNA viruses, are of limited genetic complexity and their population sizes are extremely large. Their evolution concerns complex, indeterminate mixtures of genomes rather than a single, determinate species.
DNA sequence duplications trigger gene inactivation in Neurospora crassa.
Authors: Selker E U , Garrett P W
Published: May 2006

Transforming sequences are faithfully replicated in vegetative cells of Neurospora but are typically subject at high frequency to sequence alterations and methylation in the period between fertilization and nuclear fusion. Previous work showed a correlation between the occurrence of these radical changes, referred to by the acronym RIP, and the presence of sequence duplications resulting from the introduced DNA. Various possible causes for the RIP process were investigated. Introduction of a single copy of a DNA fragment containing the Neurospora am gene into a strain having a deletion of this DNA did not lead to RIP, whereas introduction of two or more copies of the same fragment did. A conventional cross of strains having single copies of am at unlinked chromosomal locations was used to build a strain duplicated for am. Both copies of the duplicated gene were subject to the RIP process in crosses of this strain. We conclude that sequence duplications, per se, trigger RIP.
Generation of lymphokine-activated killer cells: synergy between tumor necrosis factor and interleukin 2.
Authors: Chouaib S , Bertoglio J , Blay J Y , Marchiol-Fournigault C , Fradelizi D
Published: May 2006

Large granular lymphocytes (LGL) can be activated by interleukin 2 (IL-2) to lymphokine-activated killers (LAK). The effect of tumor necrosis factor (TNF) on LAK generation was investigated. TNF was found to act synergistically with low concentrations of IL-2 (0.10-0.25 ng/ml), which were ineffective by themselves in inducing LAK activity, to promote the differentiation of LGL into non-major histocompatibility complex-restricted killers. When IL-2 was used at concentrations optimal for LAK generation, TNF did not further enhance this phenomenon. Specific binding of 125I-labeled TNF to LGL was increased by IL-2 stimulation. Scatchard analysis of TNF binding revealed the existence of two classes of binding sites with markedly different affinities (Kd values of 57 and 600 pM). We also demonstrated that the IL-2/TNF synergistic induction of LAK activity did not involve either IL-1 or interferon-gamma. This IL-2/TNF synergistic effect was blocked by anti-Tac antibodies. Immunofluorescence analysis revealed that IL-2/TNF selectively up-regulated Tac antigen expression on LAK precursors. Our results suggest a functional interaction between IL-2 and TNF on LAK precursors, which results in a reduction of the IL-2 concentration required for differentiation of LGL into LAK killers.
Sequencing of proteins from two-dimensional gels by using in situ digestion and transfer of peptides to polyvinylidene difluoride membranes: application to proteins associated with sensitization in Aplysia.
Authors: Kennedy T E , Gawinowicz M A , Barzilai A , Kandel E R , Sweatt J D
Published: May 2006

We have developed a method for obtaining partial internal amino acid sequence data from proteins isolated directly from preparative two-dimensional polyacrylamide gels. Proteins from a crude cell homogenate are separated using preparative two-dimensional polyacrylamide gel electrophoresis. Then, the gel is stained with Coomassie blue and the protein spots of interest are cut out. The in situ protein is digested with Staphylococcus aureus V8 protease in a second polyacrylamide gel and the peptides are separated by one-dimensional polyacrylamide gel electrophoresis. The peptides are then electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced using an automated gas phase sequencer. Using this method, we have obtained amino acid sequence data for two proteins that are altered after long-term sensitization: actin and Aplysia protein 407. In addition, we have obtained amino acid sequence data for rat protein 425, a protein that appears to be homologous to Aplysia protein 407.
Aldosterone increases the apical Na+ permeability of toad bladder by two different mechanisms.
Authors: Asher C , Garty H
Published: May 2006

The aldosterone-induced augmentation of Na+ transport in toad bladder was analyzed by comparing the hormonal actions on the transepithelial short-circuit current and on the amiloride-sensitive 22Na+ uptake in isolated membrane vesicles. Incubating bladders with 0.5 microM aldosterone for 3 hr evoked more than a 2-fold increase of the short-circuit current (because of the activation or insertion of apical amiloride-blockable channels) but had no effect on the amiloride-sensitive Na+ transport in apical vesicles derived from the treated tissue. A longer incubation (e.g., 6 hr) produced an additional augmentation of the short-circuit current, which was accompanied by about a 3-fold increase of the channel activity in isolated membranes. The stimulatory effect of aldosterone sustained in vesicles was inhibited by the antagonist spironolactone (present at 1000-fold excess) and the protein synthesis inhibitor cycloheximide (1 microM). In addition, triiodothyronine and butyrate, previously reported to partly inhibit the aldosterone-induced increase in short-circuit current, blocked the hormonal effect in vesicles. It is suggested that aldosterone elevates the apical Na+ permeability of target epithelia by two different mechanisms: a relatively fast effect (less than or equal to 3 hr), which is insensitive to triiodothyronine or butyrate and is not sustained by the isolated membrane, and a slower or later (greater than 3 hr) response blocked by these reagents, which is preserved by the isolated membrane. The data also indicate that these processes are mediated by different nuclear receptors.
Selenoprotein A of the clostridial glycine reductase complex: purification and amino acid sequence of the selenocysteine-containing peptide.
Authors: Sliwkowski M X , Stadtman T C
Published: May 2006

A selenium-containing protein, selenoprotein A, is an essential component of the clostridial glycine reductase complex. This enzyme complex catalyzes the reductive deamination of glycine, which is coupled to the esterification of orthophosphate resulting in the formation of ATP. Sequence information was obtained by automated Edman degradation of peptides generated by digesting carboxamidomethylated selenoprotein A with chymotrypsin or trypsin or with endoproteinase Arg-C followed by Staphylococcus aureus V8 protease. The sequence near the selenocysteine (Sec) residue is -Cys-Phe-Val-Sec-Thr-Ala-Ala-Gly-Ala-Met-Asp-Leu-Glu-Asn-Glu-Lys-. Selenium-containing peptides isolated from digests of carboxamidomethylated selenoprotein A with trypsin or endoproteinase Arg-C were found to be blocked at the amino terminus. The sequence of the selenocysteine-containing peptide from selenoprotein A shows no homology with those of two other selenoproteins, glutathione peroxidase and formate dehydrogenase.
Activation of a preexisting cellular factor as a basis for adenovirus E1A-mediated transcription control.
Authors: Reichel R , Kovesdi I , Nevins J R
Published: May 2006

Transcription of the set of early adenovirus genes is subject to positive control by the viral E1A gene. For one early viral gene, the E2 gene, this induction involves an increase in a cellular promoter-specific factor termed E2F. We have analyzed the kinetics for this induction and find that E2F is present at only very low levels in extracts of uninfected cells or cells infected for up to 3 hr with adenovirus type 5. The factor increases rapidly at 5 hr and reaches a maximal level at 7-8 hr. The kinetics of induction of the factor are thus coincident with the induction of E2 transcription. The 13S E1A gene product (289-amino acid protein), which is required for the efficient activation of E2 transcription in a productive infection, is also responsible for the activation of E2F, because infection with mutant strain pm975 (13S+, 12S-) induces the factor, whereas no increase of E2F occurs in cells infected by mutant strain dl1500 (13S-, 12S+). Finally, increase in the factor does not involve synthesis of any new protein, because extracts prepared from cells infected with adenovirus type 5 and treated with cycloheximide from 1 hr after infection contain approximately the same level of E2F as extracts from infected but untreated cells. From these results, we conclude that activation of E2F, as a posttranslational event, is responsible for the stimulation of E2 transcription by E1A.
Red light stimulates an electrogenic proton pump in Vicia guard cell protoplasts.
Authors: Serrano E E , Zeiger E , Hagiwara S
Published: May 2006

Stomatal opening in response to light has a component that matches the absorption spectrum of chlorophyll; however, the intervening sensory transduction steps are not well understood. To study this process, we illuminated Vicia faba guard cell protoplasts with red light and simultaneously recorded current flow across the plasma membrane, utilizing the patch clamp technique in the whole cell configuration. We report evidence that under voltage clamp conditions, red light (1 mmol of photons.m-2.S-1) stimulated an outward current. This response required ATP (2.5 mM) and orthophosphate (1 mM) at the cytoplasmic side of the membrane. Both red-light-stimulated currents and currents activated in the dark by the proton pump agonist fusicoccin (10 microM) were abolished by the protonophore carbonylcyanide m-chlorophenylhydrazone at 10 microM, indicating that these responses were carried by protons. Pump currents were inhibited by orthovanadate applied to the cytoplasmic side of the membrane (50% inhibition at 3.5 microM), implicating a H+ -ATPase. Elimination of the current by the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, in the presence of saturating concentrations of ATP, pointed to a requirement for photosynthetically active chloroplasts. We conclude that red light stimulates an electrogenic proton pump at the plasmalemma of Vicia guard cells and that chloroplasts modulate this response.