bluuebunny/crossref_metadata_2025 huggingface dataset:
Melittin binding causes a large calcium-dependent conformational change in calmodulin.
Authors: Kataoka M , Head J F , Seaton B A , Engelman D M
Published: May 2006

The interaction between calmodulin and its target protein is a key step in many calcium-regulated cellular functions. Melittin binds tightly to calmodulin in the presence of calcium and is a competitive inhibitor of calmodulin function. Using melittin as a model for the target peptide of calmodulin, we have found a large Ca2+-dependent conformational change of calmodulin in solution induced by peptide binding. Mg2+ does not substitute for Ca2+ in producing the conformation change. Small-angle x-ray scattering has shown that calmodulin exists as a dumbbell in solution, similar to that observed in the crystalline state. Our present measurements reveal that the overall structure of the Ca2+-calmodulin-melittin complex is not a dumbbell but a globular shape. Upon binding melittin, the radius of gyration decreases from 20.9 to 18.0 A and the largest dimension decreases from 60 to 47.5 A. In the absence of calcium, however, melittin has little effect on the solution structure of calmodulin.
Reconstitution of a bacterial periplasmic permease in proteoliposomes and demonstration of ATP hydrolysis concomitant with transport.
Authors: Bishop L , Agbayani R , Ambudkar S V , Maloney P C , Ames G F
Published: May 2006

The histidine periplasmic permease of Salmonella typhimurium has been partially purified and reconstituted into proteoliposomes. In this in vitro preparation, transport activity is completely dependent on the presence of all four permease proteins (HisJ, HisQ, HisM, and HisP) and on internal ATP. The reconstituted system shows initial rates of transport that are comparable to those obtained with right-side-out membrane vesicles and it establishes a 100-fold concentration gradient for histidine. Proteoliposomes also transport histidine when GTP replaces ATP. Proteoliposomes do not catalyze significant ATP hydrolysis until histidine transport is initiated by addition of substrate along with HisJ, the water-soluble histidine-binding protein. Both initially and throughout the course of substrate transport there is a concomitant hydrolysis of ATP, with an apparent stoichiometry (ATP/histidine) of 5:1. These experiments demonstrate directly that ATP is the source of energy for periplasmic permeases, thus resolving previous controversies on this topic.
Targeting and processing of glycophorins in murine erythroleukemia cells: use of brefeldin A as a perturbant of intracellular traffic.
Authors: Ulmer J B , Palade G E
Published: May 2006

We previously showed that glycophorins are expressed in virus-transformed, murine erythroleukemia cells; we detected four glycophorin precursors (two more than in normal erythroblasts) and found that two of them are not translocated or are inefficiently translocated across the endoplasmic reticulum (ER) membrane. By using the drug brefeldin A to block intracellular transport of proteins from the ER to the Golgi complex, the translocated precursors were shown to accumulate in the ER, while the untranslocated forms were rapidly degraded with an intracellular half-life of approximately 20 min. Brefeldin A did not inhibit the synthesis of fatty acylation of the precursors but substantially delayed their acquisition of O-linked oligosaccharides, which indicates that murine glycophorins are fatty acylated in the ER and O-glycosylated in the Golgi complex. Even after 6 hr in brefeldin A, glycophorins were only partially glycosylated, resulting in the accumulation of glycoproteins apparently sialylated but lower in apparent molecular mass than mature glycophorins. Complete glycophorin processing resumed only after removal of the drug. In murine erythroleukemia cells, brefeldin A caused a rapid and extensive disorganization of the entire Golgi complex accompanied by the accumulation of membranes in a part of the ER closely associated with ER transitional elements. These findings extend recently published results [Lippincott-Schwartz, J., Yuan, L. C., Bonifacino, J. S. & Klausner, R. D. (1989) Cell 56, 801-813] and suggest that brefeldin A induces net membrane flow from the entire Golgi complex to the ER.
Loss of distinct regions on the short arm of chromosome 17 associated with tumorigenesis of human astrocytomas.
Authors: el-Azouzi M , Chung R Y , Farmer G E , Martuza R L , Black P M , Rouleau G A , Hettlich C , Hedley-Whyte E T , Zervas N T , Panagopoulos K
Published: May 2006

Astrocytomas, including glioblastoma multiforme, represent the most frequent and deadly primary neoplasms of the human nervous system. Despite a number of previous cytogenetic and oncogene studies primarily focusing on malignant astrocytomas, the primary mechanism of tumor initiation has remained obscure. The loss or inactivation of "tumor suppressor" genes are thought to play a fundamental role in the development of many human cancers. Thus, we have analyzed astrocytomas of various histological malignancy grades with polymorphic DNA markers to search for specific chromosomal deletions potentially pointing to loci containing tumor suppressor genes. Loss of constitutional heterozygosity indicating chromosomal loss or deletions was most frequently seen for markers on the short arm of chromosome 17 in 50% of the informative tumors (5 of 10 informative cases) and, to a lesser extent, for markers on chromosomes 1 and 10. Deletions on chromosome 17p were seen in both low-grade and high-grade malignant astrocytomas, suggesting that this chromosome may contain a tumor suppressor gene associated with the early events in tumorigenesis. The common region of deletions on the short arm of chromosome 17 is, therefore, clearly distinct from the gene causing von Recklinghausen neurofibromatosis (NF1), a tumor syndrome associated with glial tumors that maps to the long arm of chromosome 17. The search for progressively smaller deletions on chromosome 17p in astrocytomas may be the way to clone and characterize this locus, thus leading to insights into normal and abnormal growth and differentiation of glial cells.
Gating of single Shaker potassium channels in Drosophila muscle and in Xenopus oocytes injected with Shaker mRNA.
Authors: Zagotta W N , Hoshi T , Aldrich R W
Published: May 2006

The voltage-dependent gating mechanism of single A-type potassium channels coded for by the Shaker locus of Drosophila was studied by single-channel recording. A-type channels expressed in Xenopus oocytes injected with Shaker B and Shaker D mRNA exhibited gating and voltage dependence that were qualitatively similar to those of the native Shaker A-types channels from embryonic myotubes. In all three channel types the molecular transition rates leading to the first opening were voltage-dependent, whereas all transitions after the first opening, including inactivation, were independent of voltage. While these channels exhibit some quantitative differences in their transition rates that account for the observed differences in macroscopic currents, in all three cases the voltage dependence of the macroscopic currents is determined by a voltage dependence in the time to first opening. This gating mechanism is similar to that of the vertebrate voltage-gated sodium channel and, together with the sequence similarities in the S4 region of the proteins, suggests a conserved mechanism for activation and inactivation.
Spontaneous electrical activity of interstitial cells of Cajal isolated from canine proximal colon.
Authors: Langton P , Ward S M , Carl A , Norell M A , Sanders K M
Published: May 2006

Interstitial cells of Cajal (ICC) have been suggested as pacemaker cells in the gastrointestinal tract. A method was developed to isolate ICC from the slow-wave pacemaker region of the canine proximal colon. These cells were identified under phase-contrast microscopy, and their identity was verified by comparing their ultrastructure with the morphology of ICC in situ. Patch-clamp experiments demonstrated that these cells are excitable; voltage-dependent inward and outward currents were elicited by depolarization. Inward current transients were identified as calcium currents. A portion of the outward current appears to be due to Ca2+-activated K channels commonly expressed in these cells. ICC were also spontaneously active, generating electrical depolarizations similar in waveform to slow-wave events of intact colonic muscles. These findings are consistent with the hypothesis that ICC initiate rhythmicity in the colon.
Geometry of a branched DNA structure in solution.
Authors: Cooper J P , Hagerman P J
Published: May 2006

An approximate geometry of a stable, four-way DNA junction has been determined in free solution by applying the technique of transient electric birefringence. The current approach consists of (i) construction of a set of six molecules in which two of the four arms of a synthetic junction are elongated by approximately 9-fold (in each of the six possible two-arm combinations), (ii) determination of the ratios of the longest birefringence decay time of each elongated junction to the decay time of a linear control molecule, and (iii) comparison of the experimental ratios with corresponding ratios computed as a function of the junction interarm angle. The result is a set of six angles that define the geometry of the junction. In the presence of magnesium ions, the junction adopts a geometry in which particular pairs of arms approach colinearity. Furthermore, the geometry of the junction is significantly altered in the absence of magnesium, adopting a more uniform structure, although such an effect is not apparent on gels. The application of transient electric birefringence, as described in the current study, should be useful for the characterization of a broad range of tertiary structures in both DNA and RNA.
Expression and nucleotide sequence of a plasmid-determined divalent cation efflux system from Alcaligenes eutrophus.
Authors: Nies D H , Nies A , Chu L , Silver S
Published: May 2006

Resistance to cobalt, zinc, and cadmium specified by the czc determinant on plasmid pMOL30 in Alcaligenes eutrophus results from a cation efflux system. Five membrane-bound polypeptides that were expressed in Escherichia coli from this determinant under the control of a phage T7 promoter were assigned to four open reading frames identified in the nucleotide sequence of the 6881-base-pair fragment containing the czc putative operon. The contributions of the polypeptides to the cation efflux system were analyzed with deletion derivatives of the 6.9-kilobase fragment, constructed, and expressed in E. coli under the control of the phage T7 promoter and in A. eutrophus under the control of the lac promoter.
Induction of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase activity by fungal elicitor in cultures of Petroselinum crispum.
Authors: McCue K F , Conn E E
Published: May 2006

The effects of a cell wall fraction of the fungus Phytophthora megasperma on the enzymatic activities of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) in extracts of cultured parsley cells (Petroselinum crispum) were examined. The specific activity of a plastidic form of DAHP synthase, designated DS-Mn by Ganson et al. [Ganson, R. J., d'Amato, T. A. & Jensen, R. A. (1986) Plant Physiol. 82, 203-210], was increased 2- to 3-fold in extracts of treated cells, with maximum induction occurring with 60 micrograms of fungal elicitor per ml after 6-8 hr. The cytosolic form of DAHP synthase, DS-Co, was unaffected by fungal elicitor. In the same experiments, phenylalanine ammonia-lyase activity (EC 4.3.1.5) increased, while no effect on isoforms of chorismate mutase (EC 5.4.99.5) was observed. The subcellular localization of the two DAHP synthase isoforms in parsley was confirmed by differential centrifugation. Prior treatment of cultures with actinomycin D or cycloheximide prevented the increase in DS-Mn activity, indicating transcriptional regulation.
Tenascin, an extracellular matrix protein, exerts immunomodulatory activities.
Authors: Rüegg C R , Chiquet-Ehrismann R , Alkan S S
Published: May 2006

Tenascin is a nonubiquitous extracellular matrix protein mainly expressed during morphogenesis in embryonal life. In adults it reappears in malignant tumors and during inflammation and tissue repair. Extracellular matrix proteins can alter cell morphology, adhesion, motility, differentiation, and growth. Since cells of the immune system can express receptors for extracellular matrix, we investigated the effects of tenascin on human monocytes and T and B lymphocytes. Tenascin inhibited monocyte adhesion to fibronectin and enhanced the LFA-1 (lymphocyte function-associated antigen 1)-dependent clustering of Epstein-Barr virus-transformed B cells. The physiological consequences of the effects of tenascin were studied in several T-cell activation models. Tenascin inhibited T-cell activation induced by a soluble antigen (tetanus toxoid), alloantigens, or the mitogen concanavalin A. However, T-cell activation with phytohemagglutinin, crosslinked anti-CD3 antibody, or a mixture of ionomycin and phorbol ester was not inhibited by tenascin. Tenascin did not prevent interleukin 2-dependent T-cell growth or the cytolytic activity of an antigen-specific CD4+ T-cell clone. These results suggest that tenascin alters the adhesion properties of human monocytes, B cells, and T cells. The in vitro immunosuppressive activity of tenascin might be due to abrogation of an accessory cell function at an early stage of the interaction between antigen-presenting cells and T cells.
Isolation and visualization of large compact ribonucleoprotein particles of specific nuclear RNAs.
Authors: Spann P , Feinerman M , Sperling J , Sperling R
Published: May 2006

We have previously shown that nuclear transcripts of the multifunctional enzyme, carbamoyl-phosphate synthetase, aspartate transcarbamylase, dihydroorotase RNA can be released from nuclei of Syrian hamster cells as compact ribonucleoprotein (RNP) particles that sediment at the 200S region in a sucrose gradient. The 200S nuclear RNP particles contain U1, U2, and U6 small nuclear RNPs, which are known to be required for splicing of pre-mRNA, as integral components of the particles. In this study we demonstrate that nuclear transcripts of dihydrofolate reductase in Syrian hamster cells and of beta-actin in both Syrian hamster and human cells are also released from the respective nuclei as 200S particles--despite the difference in length of these RNAs. Electron microscopy of the 200S particles revealed discrete compact composite structures with a cross section of approximately 50 nm. Finding that two more nuclear RNAs from two different cell types and two different species are released as 200S RNP particles suggests a general mode for packaging of heterogeneous nuclear RNA in large compact RNP particles the size of which is independent of the RNA length.
Activation of yeast RNA polymerase II transcription by a thymidine-rich upstream element in vitro.
Authors: Lue N F , Buchman A R , Kornberg R D
Published: May 2006

A thymidine-rich sequence upstream of the DED1 gene of Saccharomyces cerevisiae activated transcription of the CYC1 promoter by RNA polymerase II in vitro. Activation was inhibited by an excess of an oligonucleotide with the same but not a closely related thymidine-rich sequence, pointing to the involvement of a specific thymidine-rich element-binding factor. The extent of activation was as great as 30-fold and showed a similar distance and orientation dependence and a similar effect of deletions in vitro as in vivo.
Tissue-specific splicing mutation in acute intermittent porphyria.
Authors: Grandchamp B , Picat C , Mignotte V , Wilson J H , Te Velde K , Sandkuyl L , Roméo P H , Goossens M , Nordmann Y
Published: May 2006

An inherited deficiency of porphobilinogen deaminase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] in humans is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. It was previously demonstrated that porphobilinogen deaminase is encoded by two distinct mRNA species expressed in a tissue-specific manner. Analysis of the genomic sequences indicated that these two mRNAs are transcribed from two promoters and only differ in their first exon. The first mutation identified in the human porphobilinogen deaminase gene is a single-base substitution (G----A) in the canonical 5' splice donor site of intron 1. This mutation leads to a particular subtype of acute intermittent porphyria characterized by the restriction of the enzymatic defect to nonerythropoietic tissues. Hybridization analysis using oligonucleotide probes after in vitro amplification of genomic DNA offers another possibility of detecting asymptomatic carriers of the mutation in affected families.
De novo amplification within a "silent" human cholinesterase gene in a family subjected to prolonged exposure to organophosphorous insecticides.
Authors: Prody C A , Dreyfus P , Zamir R , Zakut H , Soreq H
Published: May 2006

A 100-fold DNA amplification in the CHE gene, coding for serum butyrylcholinesterase (BtChoEase), was found in a farmer expressing the "silent" CHE phenotype. Individuals homozygous for this gene display a defective serum BtChoEase and are particularly vulnerable to poisoning by agricultural organophosphorous insecticides, to which all members of this family had long been exposed. DNA blot hybridization with regional BtChoEase cDNA probes suggested that the amplification was most intense in regions encoding central sequences within BtChoEase cDNA, whereas distal sequences were amplified to a much lower extent. This is in agreement with the "onion skin" model, based on amplification of genes in cultured cells and primary tumors. The amplification was absent in the grandparents but present at the same extent in one of their sons and in a grandson, with similar DNA blot hybridization patterns. In situ hybridization experiments localized the amplified sequences to the long arm of chromosome 3, close to the site where we previously mapped the CHE gene. Altogether, these observations suggest that the initial amplification event occurred early in embryogenesis, spermatogenesis, or oogenesis, where the CHE gene is intensely active and where cholinergic functioning was indicated to be physiologically necessary. Our findings demonstrate a de novo amplification in apparently healthy individuals within an autosomal gene producing a target protein to an inhibitor. Its occurrence in two generations from a family under prolonged exposure to parathion indicates that organophosphorous poisons may be implicated in previously unforeseen long-term ecological effects.
Biochemical and crystallographic characterization of a complex of c-Ha-ras p21 and caged GTP with flash photolysis.
Authors: Schlichting I , Rapp G , John J , Wittinghofer A , Pai E F , Goody R S
Published: May 2006

The GTP binding domain of the c-Ha-ras protooncogene product (p21'c) and the corresponding region from an oncogenic mutant form of the protein in which glycine at position 12 has been replaced by valine [p21'(G12V)] have been crystallized with P3-1-(2-nitro)phenylethylguanosine 5'-O-triphosphate (caged GTP) at their active sites. The crystals give x-ray diffraction patterns to a resolution of better than 0.3 nm. Photolysis can be achieved in the crystal, after which GTP hydrolysis takes place at the rate expected from solution studies. Complete x-ray data sets have been obtained for the starting caged-GTP state and the final GDP state after photolysis and hydrolysis, demonstrating the feasibility of time-resolved structural investigations of the process of GTP hydrolysis.
Drosophila NK-homeobox genes.
Authors: Kim Y , Nirenberg M
Published: May 2006

Four Drosophila melanogaster homeobox genes were found by screening a genomic DNA library with oligodeoxynucleotides that correspond to a conserved amino acid sequence that is part of the putative of homeobox proteins that recognizes nucleotide sequences in DNA. The amino acid sequences of NK-2, NK-3, and NK-4 homeoboxes are more closely related to one another (59-66% homology) than they are to other Drosophila homeoboxes (28-54% homology), whereas the homeobox of NK-1 is most closely related, in order of decreasing homology, to muscle segment homeobox, zerknüllt-1, NK-3, and distal-less homeoboxes. Three of the genes, NK-1, NK-3, and NK-4, comprise a cluster of homeobox genes located in the 93E1-5 region of the right arm of the third chromosome, whereas the fourth homeobox gene, NK-2, is located in the 1C1-5 region of the X chromosome.
c-ets-2 protooncogene has mitogenic and oncogenic activity.
Authors: Seth A , Watson D K , Blair D G , Papas T S
Published: May 2006

An expression vector containing the murine c-ets-2 protooncogene cDNA was introduced into NIH 3T3 cells by DNA transfection. The cells transfected with this construct showed foci of densely growing, morphologically altered cells, when grown either in low-serum (0.05%) or in serum-free medium. The c-ets-2-derived foci contained additional copies of the c-ets-2 gene, Northern blot analysis demonstrated overexpression of a c-ets-2-specific 2.5-kilobase RNA, and ets-specific antiserum recognized a 56-kDa protein. Overexpression of the c-ets-2-encoded protein stimulated cell proliferation and abolished their serum requirement. The c-ets-2 transfected cells formed colonies in semisolid medium and induced tumors in nude mice, indicating that c-ets-2 can be a transforming gene when overexpressed in these cells. This work demonstrates that a member of the c-ets gene family has transforming and mitogenic activity. In addition, the role of c-ets-2 in cell proliferation and its location in the minimal Down syndrome region on chromosome 21 implicates its involvement in the phenotypic changes associated with Down syndrome.
Regulation of a bifunctional mRNA results in synthesis of secreted and nuclear probasin.
Authors: Spence A M , Sheppard P C , Davie J R , Matuo Y , Nishi N , McKeehan W L , Dodd J G , Matusik R J
Published: May 2006

Probasin, a rat prostatic protein, is statistically related to members of a protein family that includes serum, cellular, and nuclear proteins. In vivo, probasin appears both in the secretions and in the nucleus of prostatic epithelial cells. Using primer extension and S1 nuclease protection assays we detected only one probasin mRNA. Thus, the localization of this protein to two separate cellular regions must be encoded by this one mRNA. Furthermore, in vitro translation of synthetic probasin mRNA demonstrated that a protein containing a signal peptide and a protein lacking a signal peptide were synthesized by initiation at different AUG codons. These data are consistent with a mechanism of translational regulation of a eukaryotic bifunctional mRNA.
Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants.
Authors: Lam E , Benfey P N , Gilmartin P M , Fang R X , Chua N H
Published: May 2006

The 35S promoter of cauliflower mosaic virus (CaMV) is able to confer high-level gene expression in most organs of transgenic plants. A cellular factor from pea and tobacco leaf tissue, which recognizes nucleotides in a tandemly repeated TGACG motif at the -75 region of this promoter, has been detected by DNase I footprinting and gel retardation assays. This factor is named activation sequence factor 1 (ASF-1). A cellular factor binding to the two TGACG motifs can also be detected in tobacco root extracts. Mutations at these motifs inhibit binding of ASF-1 to the 35S promoter in vitro. When examined in transgenic tobacco, these mutations cause a 50% drop in leaf expression of the 35S promoter. In addition, these same mutations attenuate stem and root expression of the 35S promoter about 5- to 10-fold when compared to the level of expression in leaf. In contrast, mutations at two adjacent CCAAT-box-like sequences have no dramatic effect on promoter activity in vivo. A 21-base-pair element containing the two TGACG motifs is sufficient for binding of ASF-1 in vitro when inserted in a green-tissue-specific promoter. In vivo, the insertion of an ASF-1 binding site caused high levels of expression in root. Thus, a single factor binding site that is defined by site-specific mutations is shown to be sufficient to alter the expression pattern of promoters in vivo.
Mouse NIH 3T3 cells expressing human colony-stimulating factor 1 (CSF-1) receptors overgrow in serum-free medium containing human CSF-1 as their only growth factor.
Authors: Roussel M F , Sherr C J
Published: May 2006

Mouse NIH 3T3 cells expressing the human c-fms protooncogene encoding the receptor for colony-stimulating factor 1 (CSF-1) are able to proliferate in serum-free medium containing platelet-derived growth factor (PDGF), insulin, transferrin, and albumin as the only exogenous proteins. When PDGF and insulin were replaced by purified human recombinant CSF-1, the cells became spindle shaped and refractile, were no longer contact inhibited, and proliferated to high densities. Thus, transduction of the human c-fms gene into mouse fibroblasts can not only reprogram their growth factor requirements but can also induce ligand-dependent features of cell transformation. NIH 3T3 cells stably transformed by the feline v-fms oncogene or by a mutated, oncogenic human c-fms gene were able to proliferate in the absence of exogenous growth factors. A monoclonal antibody that prevents signal transduction by the human CSF-1 receptor inhibited the growth of cells transformed by the activated c-fms oncogene, confirming that CSF-1 receptor function was required to abrogate growth factor requirements and to maintain the transformed state.
Traveling waves of in vitro evolving RNA.
Authors: Bauer G J , McCaskill J S , Otten H
Published: May 2006

Populations of short self-replicating RNA variants have been confined to one side of a reaction-diffusion traveling wave front propagating along thin capillary tubes containing the Q beta viral enzyme. The propagation speed is accurately measurable with a magnitude of about 1 micron/sec, and the wave persists for hundreds of generations (of duration less than 1 min). Evolution of RNA occurs in the wavefront, as established by front velocity changes and gel electrophoresis of samples drawn from along the capillary. The high population numbers (approximately equal to 10(11], their well-characterized biochemistry, their short generation time, and the constant conditions make the system ideal for evolution experiments. Growth is monitored continuously by excitation of an added RNA-sensitive fluorescent dye, ethidium bromide. An analytic expression for the front velocity is derived for the multicomponent kinetic scheme that reduces, for a high RNA-enzyme binding constant, to the Fisher form v = 2 square root of kappa D, where D is the diffusion constant of the complex and kappa is the low-concentration overall replication rate coefficient. The latter is confirmed as the selective value-determining parameter by numerical solution of a two-species system.
alar4, a constitutive mutant of the A system for amino acid transport, has increased abundance of the Na+,K+-ATPase and mRNA for alpha 1 subunit of this enzyme.
Authors: Qian N X , Jones M , McDonough A , Englesberg E
Published: May 2006

A constitutive mutant, alar4, for the A system of amino acid transport, has increased activity and amount of the A system. This is accompanied by increased sensitivity to ouabain, as measured by efficiency of plating, and increased activity and abundance of the Na+,K+-ATPase that is present in the parental cell line, CHO-K1 (wild type). The latter was shown by increases in (i) ouabain-inhibitable 86Rb uptake in intact cells, (ii) ouabain-inhibitable ATPase activity in mixed membrane vesicles, and (iii) number of ouabain-binding sites and by similar Kd values for ouabain binding and K1/2 for ouabain inhibition of Na+,K+-ATPase as compared to the wild type. The increase in abundance of the Na+ pump is associated with a 4-fold increase in abundance of the mRNA for the alpha 1 subunit of the Na+,K+-ATPase. We could not detect mRNA for alpha 2 or alpha 3 or for the beta subunits. The increase in abundance of the A system and Na+,K+-ATPase is associated with a negligible increase in intracellular Na+ concentration. We propose that the increase in the abundance of the A system and the Na+,K+-ATPase is the result of a mutation in regulatory gene R1 that controls the A system and the Na+,K+-ATPase and is not due to a primary effect of a possible initial increase in Na+ concentration.
Interleukin 6 induces secretion of IgG1 by coordinated transcriptional activation and differential mRNA accumulation.
Authors: Raynal M C , Liu Z Y , Hirano T , Mayer L , Kishimoto T , Chen-Kiang S
Published: May 2006

The molecular mechanism by which interleukin 6 (IL-6) induces terminal differentiation of B cells was investigated in a subpopulation of the clonal human B-lymphoblastoid cell line CESS selected for high density of cell surface IgG1. Induction of CESS cells with IL-6 resulted in a 15-fold preferential accumulation of secreted-specific gamma 1 (gamma 1s) mRNA but not of the alternatively processed membrane-specific gamma 1 (gamma 1m) mRNA. Similarly, microseconds mRNA but not the microns mRNA of the nonproductively rearranged mu heavy-chain allele was also increased. Accompanying the differential accumulation of gamma 1s mRNA was a 4.5-fold increase in lambda light-chain mRNA, leading to secretion of IgG1. Analyses of transcription in isolated nuclei demonstrated that transcriptional activation was the primary mechanism for quantitative increase of immunoglobulin mRNAs (5.5-fold for gamma 1 and mu and at least 2-fold for lambda). Since polymerase loading is diminished by 75% before reaching the downstream gamma 1m polyadenylylation site in CESS cells, irrespective of IL-6 induction, transcriptional pausing/termination appears intrinsic and contributes to the selection of gamma 1s and gamma 1m polyadenylylation sites in activated B cells. Furthermore, differential mRNA stabilization is likely to contribute to the alteration of the gamma 1s/gamma 1m mRNA ratio at IL-6 induction.
Dopamine- and cAMP-regulated phosphoprotein (DARPP-32) and dopamine DA1 agonist-sensitive Na+,K+-ATPase in renal tubule cells.
Authors: Meister B , Fryckstedt J , Schalling M , Cortés R , Hökfelt T , Aperia A , Hemmings H C , Nairn A C , Ehrlich M , Greengard P
Published: May 2006

The cellular localization of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 that appears to mediate certain actions of dopamine in the mammalian brain by acting as an inhibitor of protein phosphatase 1, was studied in the kidney of several species. DARPP-32 mRNA and DARPP-32-like immunoreactivity were found in the cytoplasm of cells in the thick ascending limb of the loop of Henle. The specific dopamine DA1 agonist SKF 82526 caused a dose-dependent inhibition of Na+,K+-ATPase activity, which could be blocked by SCH 23390, a specific DA1 antagonist, and by PKI-(5-24) amide, a specific inhibitor of cAMP-dependent protein kinase. The results indicate that DA1 dopamine receptors and DARPP-32, an intracellular third messenger for dopamine, are part of the signal-transduction process for dopamine acting on renal tubule cells.
Discrimination of heterogenous mRNAs encoding strychnine-sensitive glycine receptors in Xenopus oocytes by antisense oligonucleotides.
Authors: Akagi H , Patton D E , Miledi R
Published: May 2006

Three synthetic oligodeoxynucleotides complementary to different parts of an RNA encoding a glycine receptor subunit were used to discriminate heterogenous mRNAs coding for glycine receptors in adult and neonatal rat spinal cord. Injection of the three antisense oligonucleotides into Xenopus oocytes specifically inhibited the expression of glycine receptors by adult spinal cord mRNA. In contrast, the antisense oligonucleotides were much less potent in inhibiting the expression of glycine receptors encoded by neonatal spinal cord mRNA. Northern blot analysis revealed that the oligonucleotides hybridized mostly to an adult cord transcript of approximately 10 kilobases in size. This band was also present in neonatal spinal cord mRNA but its density was about one-fourth of the adult cord message. There was no intense band in the low molecular weight position (approximately 2 kilobases), the existence of which was expected from electrophysiological studies with size-fractionated mRNA of neonatal spinal cord. Our results suggest that in the rat spinal cord there are at least three different types of mRNAs encoding functional strychnine-sensitive glycine receptors.
Independent expression of human alpha or beta platelet-derived growth factor receptor cDNAs in a naive hematopoietic cell leads to functional coupling with mitogenic and chemotactic signaling pathways.
Authors: Matsui T , Pierce J H , Fleming T P , Greenberger J S , LaRochelle W J , Ruggiero M , Aaronson S A
Published: May 2006

Distinct genes encode alpha and beta platelet-derived growth factor (PDGF) receptors that differ in their abilities to be triggered by three dimeric forms of the PDGF molecule. We show that PDGF-receptor mitogenic function can be reconstituted in a naive hematopoietic cell line by introduction of expression vectors for either alpha or beta PDGF receptor cDNAs. Thus, each receptor is independently capable of coupling with mitogenic signal-transduction pathways inherently present in these cells. Activation of either receptor also resulted in chemotaxis, alterations in inositol lipid metabolism, and mobilization of intracellular Ca2+. The magnitude of these functional responses correlated well with the binding properties of the different PDGF isoforms to each receptor. Thus, availability of specific PDGF isoforms and relative expression of each PDGF-receptor gene product are major determinants of the spectrum of known PDGF responses.
Tumor necrosis factor induces phosphorylation of a 28-kDa mRNA cap-binding protein in human cervical carcinoma cells.
Authors: Marino M W , Pfeffer L M , Guidon P T , Donner D B
Published: May 2006

Tumor necrosis factor alpha (TNF-alpha) stimulated the phosphorylation of a 28-kDa protein (p28) in the ME-180 line of human cervical carcinoma cells. The effect of TNF-alpha on the phosphorylation state of p28 was rapid (4-fold increase within 15 min) and persistent, remaining above the basal level for at least 2 hr. The specific binding of 125I-labeled TNF-alpha to cell-surface binding sites, the stimulation of p28 phosphorylation by TNF-alpha, and the inhibition of cell proliferation by TNF-alpha occurred with nearly identical dose-response relationships. Two-dimensional SDS/PAGE resolved p28 into two isoforms having pI values of 6.2 and 6.1. A phosphorylated cap-binding protein was substantially enriched from lysates of control or TNF-alpha-treated ME-180 cells by affinity chromatography with 7-methylguanosine 5'-triphosphate-Sepharose. The phosphoprotein recovered from this procedure was the substrate for TNF-alpha-promoted phosphorylation, p28. Thus, TNF-alpha stimulates the phosphorylation of this mRNA cap-binding protein, which may be involved in the transduction of TNF-alpha-receptor binding into cellular responses.
Specific binding of angiogenin to calf pulmonary artery endothelial cells.
Authors: Badet J , Soncin F , Guitton J D , Lamare O , Cartwright T , Barritault D
Published: May 2006

Specific binding of angiogenin (ANG) to calf pulmonary artery endothelial cells was demonstrated. Cellular binding at 4 degrees C of 125I-labeled human recombinant ANG was time and concentration dependent, reversible, and saturable in the presence of increasing amounts of the unlabeled molecules. The interaction was shown to be specific since a large excess of unlabeled ANG reduced labeled ANG binding by 80%, whereas similar doses of RNase A, a structurally related protein, had no effect. Scatchard analyses of binding data revealed two apparent components. High-affinity sites with an apparent dissociation constant of 5 x 10(-9) M were shown to represent cell-specific interactions. The second component, comprising low-affinity/high-capacity sites with an apparent dissociation constant of 0.2 x 10(-6) M, was essentially associated with pericellular components. High-affinity ANG binding sites varied with cell density and were found on other endothelial cells from bovine aorta, cornea, and adrenal cortex capillary but not on Chinese hamster lung fibroblasts. Divalent copper, a modulator of angiogenesis, was found to induce a severalfold increase in specific cell-bound radioactivity. Placental ribonuclease inhibitor, a tight-binding inhibitor of both ribonucleolytic and angiogenic activities of ANG, abolished 125I-labeled human recombinant ANG binding only in the absence of copper.
AbrB, a regulator of gene expression in Bacillus, interacts with the transcription initiation regions of a sporulation gene and an antibiotic biosynthesis gene.
Authors: Robertson J B , Gocht M , Marahiel M A , Zuber P
Published: May 2006

The abrB gene of Bacillus subtilis is believed to encode a repressor that controls the expression of genes involved in starvation-induced processes such as sporulation and the production of antibiotics and degradative enzymes. Two such genes, spoVG, a sporulation gene of B. subtilis, and tycA, which encodes tyrocidine synthetase I of the tyrocidine biosynthetic pathway in Bacillus brevis, are negatively regulated by abrB in B. subtilis. To examine the role of abrB in the repression of gene transcription, the AbrB protein was purified and then tested for its ability to bind to spoVG and tycA promoter DNA. In a gel mobility shift experiment, AbrB was found to bind to a DNA fragment containing the sequence from -95 to +61 of spoVG. AbrB protein exhibited reduced affinity for DNA of two mutant forms of the spoVG promoter that had been shown to be insensitive to abrB-dependent repression in vivo. These studies showed that an upstream A + T-rich sequence from -37 to -95 was required for optimal AbrB binding. AbrB protein was also observed to bind to the tycA gene within a region between the transcription start site and the tycA coding sequence as well as to a region containing the putative tycA promoter. These findings reinforce the hypothesis that AbrB represses gene expression through its direct interaction with the transcription initiation regions of genes under its control.
Male-enhanced antigen gene is phylogenetically conserved and expressed at late stages of spermatogenesis.
Authors: Lau Y F , Chan K M , Sparkes R
Published: May 2006

The male-enhanced antigen gene (Mea) was previously isolated from a mouse testicular cDNA library by using a pool of specific antisera against the serological H-Y antigen. The present studies characterize the human and mouse cDNAs and indicate that the MEA gene is conserved at both nucleic acid and protein levels. The corresponding mRNA encodes proteins of 18-20 kDa. The phylogenetic conservation could be extended to other mammalian species by Southern blot analysis. Although the Mea gene was transcribed as a 1-kilobase mRNA in most tissues, it was expressed at the highest level in adult testis. The testis-enhanced expression of the Mea gene was associated with germ cell development at late stages of spermatogenesis. Chromosome walking experiments identified two linked genes, A and B, located within 38 kilobases of human genomic sequence. Like the MEA gene, genes A and B were coordinately transcribed in the testis, which suggests that MEA and genes A and B are members of a gene family. In situ hybridization studies localized the MEA gene to the short arm of human chromosome 6 at band p21.1-21.3, close to the major histocompatibility complex locus. The genetic conservation and testis-specific expression of the MEA gene support the hypothesis that it plays an important role in mammalian spermatogenesis and/or testis development.
Molecular studies of deletions at the human steroid sulfatase locus.
Authors: Shapiro L J , Yen P , Pomerantz D , Martin E , Rolewic L , Mohandas T
Published: May 2006

The human steroid sulfatase gene (STS) is located on the distal X chromosome short arm close to the pseudoautosomal region but in a segment of DNA that is unique to the X chromosome. In contrast to most X chromosome-encoded genes, STS expression is not extinguished during the process of X chromosome inactivation. Deficiency of STS (steryl-sulfatase; steryl-sulfate sulfohydrolase, EC 3.1.6.2) activity produces the syndrome of X chromosome-linked ichthyosis, which is one of the most common inborn errors of metabolism in man. Approximately 90% of STS- individuals have large deletions at the STS locus. We and others have found that the end points of such deletions are heterogeneous in their location. One recently ascertained subject was observed to have a 40-kilobase deletion that is entirely intragenic, permitting the cloning and sequencing of the deletion junction. Studies of this patient and of other X chromosome sequences in other subjects permit some insight into the mechanism(s) responsible for generating frequent deletions on the short arm of the X chromosome.
Selective loss of cholinergic neurons in the ventral striatum of patients with Alzheimer disease.
Authors: Lehéricy S , Hirsch E C , Cervera P , Hersh L B , Hauw J J , Ruberg M , Agid Y
Published: May 2006

Cholinergic neurons were studied by immunohistochemistry with an antiserum against human choline acetyltransferase in the caudate nucleus, putamen, and ventral striatum (including the nucleus accumbens) of three patients with Alzheimer disease and three control subjects. Immunoreactive cell bodies were mapped and counted. In the ventral striatum of patients with Alzheimer disease, a 60% decrease in the number of cholinergic neurons was observed, whereas in the caudate nucleus and putamen values for control subjects and patients were similar. To determine whether all neurons in the ventral striatum were affected, neuropeptide Y-containing neurons were also immunostained, mapped, and counted. The number of these neurons was the same in control subjects and patients with Alzheimer disease, indicating that neuronal loss is not generalized in the ventral striatum and may be specific to the cholinergic population.
Icosahedral quasicrystals of intermetallic compounds are icosahedral twins of cubic crystals of three kinds, consisting of large (about 5000 atoms) icosahedral complexes in either a cubic body-centered or a cubic face-centered arrangement or smaller (about 1350 atoms) icosahedral complexes in the β-tungsten arrangement
Authors: Pauling Linus
Published: May 2006

The twofold-axis electron-diffraction photographs of icosahedral quasicrystals are of three kinds, reflecting three different structures of the cubic crystals that by icosahedral twinning form the quasicrystals. The first kind, represented by Al
13
Cu
4
Fe
3
, contains two very large icosahedral complexes, each of about 4680 atoms, in the body-centered arrangement, with six smaller icosahedral complexes (104 atoms each) in the principal interstices. The second kind, represented by Al
5
Mn, contains four of the very large complexes in the face-centered arrangement (cubic close packing), with four of the smaller clusters in the interstices. The third kind, represented by Al
6
CuLi
3
, contains eight icosahedral complexes, each of about 1350 atoms, in the β-W arrangement. The supporting evidence for these cubic structures is discussed as well as other evidence showing that the simple quasicrystal theory, which states that quasicrystals do not involve any translational identity operations, has to be modified.
Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme.
Authors: Kasho V N , Boyer P D
Published: May 2006

Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F1,F0-ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F1,F0-ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and Pi as medium ATP concentration was lowered was determined by 18O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the Pi formed as ATP concentration is decreased to the micromolar range. The F1,F0-ATPase from neurospora mitochondria showed an even more pronounced modulation, similar to that of other F1-type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F1,F0-ATPases.
Photolysis of a photolabile precursor of ATP (caged ATP) induces microsecond ratational motions of myosin heads bound to actin.
Authors: Berger C L , Svensson E C , Thomas D D
Published: May 2006

To test the proposal that ATPase activity is coupled to the rotation of muscle cross-bridges (myosin heads attached to actin), we have used saturation-transfer EPR to detect the rotational motion of spin-labeled myosin heads (subfragment 1; S1) bound to actin following the photolysis of caged ATP (a photoactivatable analog of ATP). In order to ensure that most of the heads were bound to actin in the presence of ATP, solutions contained high (200 microns) actin concentrations and were of low (36 mM) ionic strength. Sedimentation measurements indicated that 52 +/- 2% of the spin-labeled heads were attached in the steady state of ATP hydrolysis during EPR measurements. Five millimolar caged ATP was added to the actin-S1 solution in an EPR cell in the dark, with no effect on the intense saturation-transfer EPR signal, implying a rigid actin-S1 complex. A laser pulse produced 1 mM ATP, which decreased the signal rapidly to a brief steady-state level that indicated only slightly less rotational mobility than that of free heads. After correcting for the fraction of free heads, we conclude that the bound heads have an effective rotational correlation time of 1.0 +/- 0.3 microseconds, which is about 100 times shorter (faster) than that in the absence of ATP. To our knowledge, this is the first direct evidence that myosin heads undergo rotational motion when bound to actin during the ATPase cycle. It is likely that similar cross-bridge rotations occur during muscle contraction.
Identification and characterization of a nuclear localization sequence-binding protein in yeast.
Authors: Lee W C , Mélèse T
Published: May 2006

Nuclear proteins contain specific regions that are required for entry into the nucleus. Using ligand blotting, we have shown that a 67-kDa yeast nuclear envelope protein (p67) recognizes synthetic peptides containing the yeast histone H2B or simian virus 40 large tumor antigen nuclear localization sequence. Both free peptide and peptide conjugated to human serum albumin are recognized. The interaction between p67 and the nuclear localization sequences is specific; neither a mutant peptide that is incompetent for nuclear transport in vivo nor HSA can interact with p67 on blots. Moreover, although the wild-type peptide competes for binding to p67, the mutant peptides do not. p67 appears to be located at the nuclear envelope and is not present in other subcellular fractions. The nuclear localization sequence-binding protein is not extracted from the nuclear envelope with nonionic detergents and only partially extracted with high-salt buffer or 8 M urea, suggestive of a tight association with the nuclear envelope. Together our results are consistent with a role for p67 in nuclear transport.