bluuebunny/crossref_metadata_2025 huggingface dataset:
Retinoic acid stimulates neurite outgrowth in the amphibian spinal cord.
Authors: Hunter K , Maden M , Summerbell D , Eriksson U , Holder N
Published: May 2006

There is increasing evidence that retinoic acid (RA), a vitamin A metabolite, plays a role in the development of the nervous system. Here we specifically test this notion by examining the effect of RA on neurite outgrowth from explanted segments of the axolotl spinal cord. We show that there is a threshold concentration in the region of 0.1-1 nM above which neurite outgrowth is stimulated 4-5 fold. Retinol, by contrast, only stimulated the migration of glial cells from the explants. Using HPLC we demonstrate that RA and retinol are present endogenously in the axolotl spinal cord. In addition, we have identified by immunocytochemistry with antipeptide antibodies the cells of the spinal cord that contain the binding proteins for RA (cellular RA-binding protein; CRABP) and retinol (cellular retinol-binding protein; CRBP). CRABP is found in the axons and CRBP is found in the ependyma and glial cells. These results provide strong evidence for a role for RA in the developing nervous system, and we propose a specific hypothesis involving CRBP, CRABP, retinol, and RA in the control of axon outgrowth in the spinal cord.
Differential signal transduction via T-cell receptor CD3 zeta 2, CD3 zeta-eta, and CD3 eta 2 isoforms.
Authors: Bauer A , McConkey D J , Howard F D , Clayton L K , Novick D , Koyasu S , Reinherz E L
Published: May 2006

The T-cell antigen receptor (TCR) consists of an antigen-binding heterodimer, termed Ti, which is noncovalently associated with the invariant CD3 subunits (gamma, delta, epsilon, zeta, and eta). The CD3 zeta and -eta subunits form either homodimeric or heterodimeric structures in turn associated with the other components of the TCR complex. This feature increases the structural complexity of TCRs by creating "isoforms." Both CD3 zeta and -eta are thought to play an important role in signal transduction triggered by antigen/major histocompatibility complex. To compare signaling functions of TCR isoforms, MA5.8, a CD3 zeta-eta- variant of the cytochrome c-specific, I-Ek-restricted T-cell hybridoma 2B4.11, was stably transfected with cDNAs encoding CD3 zeta and/or CD3 eta, and resulting clones were characterized. The findings indicate that signals inducing Ca2+ mobilization, phosphatidylinositol turnover, and interleukin 2 production are each transmitted by the above TCR isoforms. In contrast, tyrosine phosphorylation of the CD3 zeta subunit but not the CD3 eta subunit follows TCR stimulation. Given the general importance of tyrosine phosphorylation for receptor signaling, it is likely that this difference between TCR isoforms plays a regulatory role in T-lineage function by qualitatively or quantitatively altering signaling events.
Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.
Authors: Jiang M , Pandey S , Tran V T , Fong H K
Published: May 2006

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensitive events. Other G alpha-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to Gi-2 alpha and Gs alpha protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G alpha gene family, at least two other G alpha subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G alpha subunits were isolated and characterized. The results indicate that this G alpha subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.
Theoretical analysis of a physical mapping strategy using random single-copy landmarks.
Authors: Barillot E , Dausset J , Cohen D
Published: May 2006

An approach to physical mapping is analyzed here. This procedure consists of fingerprinting random clones with single-copy landmarks extracted randomly from a region of interest. Results are presented in terms of number of contigs (sets of overlapping clones), number of isolated clones, average length of a contig, and coverage of the genome by contigs larger than a given size. (i) The expected results of an ideal project are presented. (ii) Certain problems that could influence progress of the map (variability in clone insert length, double inserts, etc.) are considered. (iii) An optimal project, which consists of a 7-fold representative library fingerprinted with an average of five sequence-tagged sites per clone, is analyzed. (iv) We present strategical considerations for the proposed approach, and a strategy that minimizes the number of laboratory tests without significant information loss is proposed: clones are arranged on a matrix and pooled according to rows and columns. A fingerprint is determined for each pool, and analysis allows retrieval of the positive clones. This method reduces the number of laboratory tests done by a factor of 160.
Yeast artificial chromosomes spanning 8 megabases and 10-15 centimorgans of human cytogenetic band Xq26.
Authors: Little R D , Pilia G , Johnson S , D'Urso M , Schlessinger D
Published: May 2006

A successful test is reported to generate long-range contiguous coverage of DNA from a human cytogenetic band in overlapping yeast artificial chromosomes (YACs). Seed YACs in band Xq26 were recovered from a targeted library of clones from Xq24-q28 with 14 probes, including probes for the hypoxanthine guanine phosphoribosyltransferase- and coagulation factor IX-encoding genes and nine probes used in linkage mapping. Neighboring YACs were then identified by 25 "walking" steps with end-clones, and the content of 71 probes in cognate YACs was verified by further hybridization analyses. The resultant contig extends across 8 million base pairs, including most of band Xq26, with an order of markers consistent with linkage data. YAC-based mapping, thus, permits steps toward a fully integrated physical and genetic map and is probably adequate to sustain most of the human genome project.
Viral proteins associated with the Epstein-Barr virus transactivator, ZEBRA.
Authors: Katz D A , Baumann R P , Sun R , Kolman J L , Taylor N , Miller G
Published: May 2006

The BamHI Z Epstein-Barr replication activator (ZEBRA) mediates disruption of latency and induction of Epstein-Barr virus (EBV) early gene expression in latently infected lymphocytes. Polyclonal rabbit sera raised against ZEBRA were used to immunoprecipitate ZEBRA-associated proteins (ZAPs). ZAPs of 19, 21, 23, and 42 kDa were coimmunoprecipitated with ZEBRA from extracts of EBV-producing lymphoid cell lines. ZAPs were not recognized directly by the rabbit sera, but they were antigenic for EBV+ human sera. Immunoprecipitation of ZAPs by ZEBRA-specific antisera required the presence of ZEBRA. ZAPs were not coprecipitated with ZEBRA from mouse cells expressing only ZEBRA, from Raji (a cell line in which EBV is unable to complete lytic replication), or from cells treated with inhibitors of viral DNA synthesis. Thus, ZAPs are late EBV-encoded proteins. ZEBRA and ZAPs colocalized to a salt-insoluble nuclear fraction, and both were found extracellularly in crude preparations of virions. ZAPs might function to affect the cellular localization of ZEBRA, to alter its capacity to transactivate, or to influence its target gene specificity.
Chemical implementation of finite-state machines.
Authors: Hjelmfelt A , Weinberger E D , Ross J
Published: May 2006

With methods developed in a prior article on the chemical kinetic implementation of a McCulloch-Pitts neuron, connections among neurons, logic gates, and a clocking mechanism, we construct examples of clocked finite-state machines. These machines include a binary decoder, a binary adder, and a stack memory. An example of the operation of the binary adder is given, and the chemical concentrations corresponding to the state of each chemical neuron are followed in time. Using these methods, we can, in principle, construct a universal Turing machine, and these chemical networks inherit the halting problem
Urate-null rosy mutants of Drosophila melanogaster are hypersensitive to oxygen stress.
Authors: Hilliker A J , Duyf B , Evans D , Phillips J P
Published: May 2006

It has been proposed that uric acid is an important scavenger of deleterious oxygen radicals in biological systems [Ames, B. N., Cathcart, R., Schwiers, E. & Hochstein, P. (1981) Proc. Natl. Acad. Sci. USA 78, 6858-6852]. We report here an in vivo investigation of the oxygen defense role of uric acid through an analysis of mutants of the rosy (ry) gene of Drosophila melanogaster. The ry gene is the structural gene for the molybdoenzyme, xanthine dehydrogenase; xanthine dehydrogenase-null ry mutants are therefore unable to synthesize urate. The rationale of our approach was to measure the response of urate-null ry mutants to extraordinary oxygen stress as imposed by exposure to radical-generating agents and as conferred by a genetic defect in superoxide dismutase, an established oxygen defense function. We show that urate-null mutants of the ry locus are hypersensitive to paraquat, ionizing radiation, and hyperoxia. Furthermore, compound mutants doubly deficient for uric acid and Cu/Zn-containing superoxide dismutase are synthetic lethals, which are unable to complete metamorphosis under normal growth conditions. These experiments demonstrate unambiguously the importance of urate in oxygen defense in vivo and support our earlier proposal that the molybdoenzyme genetic system plays a critical role in oxygen defense in Drosophila. They also form the basis for our proposal that metamorphosis in Drosophila imposes a crisis of oxygen stress on the developing imago against which uric acid plays an important organ-specific defense. Finally, the results provide a basis for understanding the syndrome of phenotypes, including the hallmark dull brown eye color, which characterizes mutants of this classic genetic system of Drosophila.
Amino acid substitutions in the sixth transmembrane domain of P-glycoprotein alter multidrug resistance.
Authors: Devine S E , Ling V , Melera P W
Published: May 2006

Eukaryotic cells can display resistance to a wide range of natural-product chemotheraputic agents by the expression of P-glycoprotein (pgp), a putative plasma membrane transporter that is thought to mediate the efflux of these agents from cells. We have identified, in cells selected for multidrug resistance with actinomycin D, a mutant form of pgp that contains two amino acid substitutions within the putative sixth transmembrane domain. In transfection experiments, this altered pgp confers a cross-resistance phenotype that is altered significantly from that conferred by the normal protein, displaying maximal resistance to actinomycin D. These results strongly implicate the sixth transmembrane domain in the mechanism of pgp drug recognition and efflux. Moreover, they indicate a close functional homology between pgp and the cystic fibrosis transmembrane regulator in which the sixth transmembrane domain has also been shown to influence substrate specificity.
Peptide ligands for a sugar-binding protein isolated from a random peptide library.
Authors: Oldenburg K R , Loganathan D , Goldstein I J , Schultz P G , Gallop M A
Published: May 2006

Peptide ligands for the carbohydrate-binding protein concanavalin A (Con A) have been identified by screening a large, diverse peptide library expressed on the surface of filamentous phage. A dodecapeptide containing the consensus sequence Tyr-Pro-Tyr was found to bind Con A with an affinity (dissociation constant, Kd) of 46 microM, comparable to that of a known carbohydrate ligand, methyl alpha-D-mannopyranoside (Kd of 89 microM). In addition the peptide inhibited precipitation of the alpha-glucan dextran 1355 by Con A. Given the complexity of oligosaccharide synthesis, the prospect of finding peptides that competitively inhibit carbohydrate-specific receptors may simplify the development of new therapeutic agents.
CD14 is involved in control of human immunodeficiency virus type 1 expression in latently infected cells by lipopolysaccharide.
Authors: Bagasra O , Wright S D , Seshamma T , Oakes J W , Pomerantz R J
Published: May 2006

Lipopolysaccharide (LPS) potently stimulates human immunodeficiency virus type 1 (HIV-1) long terminal repeat-directed transcription in transfected monocyte-macrophage cell lines and dramatically increases HIV-1 production in the latently infected monocyte-macrophage-like cell line U1. This response to LPS, however, can only be observed after pretreatment of the U1 cells with granulocyte-macrophage colony-stimulating factor (GM-CSF). CD14, the differentiation antigen that acts as a receptor for complexes of LPS and LPS-binding protein, is now demonstrated to be involved in LPS-induced stimulation of HIV-1 replication. CD14 is shown to be expressed on a subpopulation of U1 cells only after treatment with GM-CSF and correlates with HIV-1 production stimulated by LPS. Importantly, only those U1 cells that express CD14 can be induced by LPS to upregulate HIV-1 production. In addition, a monoclonal antibody directed against CD14 can block LPS-induced stimulation of HIV-1 production from these latently infected cells.
Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers.
Authors: De Rocquigny H , Gabus C , Vincent A , Fournié-Zaluski M C , Roques B , Darlix J L
Published: May 2006

The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA annealing activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first finger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and annealing activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents.
Interferon alpha induces the expression of retinoblastoma gene product in human Burkitt lymphoma Daudi cells: role in growth regulation.
Authors: Kumar R , Atlas I
Published: May 2006

Interferon alpha (IFN-alpha) is a regulatory secretory protein with distinctive biological effects such as antiproliferative actions against many tumor cell lines, including human Burkitt lymphoma Daudi cells. The mechanism underlying growth inhibition by IFN-alpha is not well established. The growth of many mammalian cell types is also regulated by tumor suppressor retinoblastoma (RB) gene product, the RB protein. In the studies presented here, we explored the possible involvement of RB protein in the growth inhibitory action of IFN-alpha in the Daudi cell model system. We observed that IFN-alpha induces a 3- to 10-fold increased expression of RB protein in growth-sensitive Daudi cells but not in the growth-resistant variant of Daudi cells. IFN-alpha-mediated induction of RB protein was an early event that preceded the period of growth inhibition of Daudi cells. IFN-alpha-induced RB protein predominantly exists as the underphosphorylated form. Addition of antibody against IFN-alpha to Daudi cells resulted in the inhibition of constitutive expression of RB protein and stimulation of [3H]thymidine incorporation. These results demonstrate that the induction of RB protein expression in IFN-alpha-treated Daudi cells could constitute an important mechanism of IFN-alpha-mediated growth regulation.
The crtE gene in Erwinia herbicola encodes geranylgeranyl diphosphate synthase.
Authors: Math S K , Hearst J E , Poulter C D
Published: May 2006

A cluster of genes essential for the biosynthesis of carotenoids in Erwinia herbicola has been isolated and characterized [Armstrong, G.A., Alberti, M. & Hearst, J. E. (1990) Proc. Natl. Acad. Sci. USA 87, 9975-9979]. Related gene clusters are found in other carotenoid-producing bacteria. Two of these genes, crtB and crtE, have been assigned to enzymes responsible for conversion of geranylgeranyl diphosphate (GGPP) to prephytoene diphosphate and prephytoene diphosphate to phytoene, respectively. We isolated crtE from the Er. herbicola cluster by PCR amplification and cloned the coding region into the Escherichia coli expression vector pARC306N. Es. coli JM101 was transformed with the expression plasmid, and transformants were assayed for GGPP synthase and phytoene synthase activity. Extracts from JM101/pSM145 accumulated [14C]GGPP when incubated with [14C]isopentenyl diphosphate and farnesyl diphosphate, whereas similar incubations with [3H]GGPP did not yield prephytoene diphosphate or phytoene. Thus, crtE encodes GGPP synthase.
Yeast glycoprotein biosynthesis: MNT1 encodes an alpha-1,2-mannosyltransferase involved in O-glycosylation.
Authors: Häusler A , Ballou L , Ballou C E , Robbins P W
Published: May 2006

The Saccharomyces cerevisiae MNT1 gene encodes a Golgi mannosyltransferase. Gene disruption of the MNT1 locus leads to a greater than 90% reduction of specific alpha-1,2-mannosyltransferase activity with alpha-methylmannoside as acceptor. Null mutants of MNT1 are viable, have no apparent growth defect, and are blocked in the elongation of protein O-linked mannobiose. Structural analysis of the N-linked outer chain isolated from an mnn1 mnn10 mnt1 strain revealed no alteration in carbohydrate structure compared to the parental mnn1 mnn10 strain. The MNT1 gene is identical to KRE2, and mutations in the gene render cells resistant to the killer toxin K1 of S. cerevisiae, which suggests a role for O-mannosylated proteins in the resistance mechanism. In addition, MNT1 is part of a multigene family whose members are presumed to be yeast Golgi mannosyltransferases.
"Rogue" lymphocytes among Ukrainians not exposed to radioactive fall-out from the Chernobyl accident: the possible role of this phenomenon in oncogenesis, teratogenesis, and mutagenesis.
Authors: Neel J V , Awa A A , Kodama Y , Nakano M , Mabuchi K
Published: May 2006

Cultured lymphocytes exhibiting extreme cytogenetic damage (rogue cells) were observed in preparations from 8 of 24 individuals sampled in Krasilovka, a Ukrainian village receiving little or no increased radiation after the Chernobyl disaster, but were not observed in an additional 24 persons from two Russian towns in the more contaminated area. This observation cements the worldwide occurrence of these cells. The present data plus a review of the literature establish that rogue cells appear in brief bursts simultaneously in certain individuals of discrete populations. We suggest that the pattern is consistent with the action of a viral trigger that acts directly or indirectly--the latter possibly through the activation of latent chromosomal retroposons. If this phenomenon occurs in other tissues, it may have important implications for oncogenesis, teratogenesis, mutagenesis, and evolution.
Paleozoic record of morphologica diversity in blastozoan echinoderms.
Authors: Foote M
Published: May 2006

There has been extensive debate about the magnitude and implications of morphological diversity in early Paleozoic animals, with some workers using apparently rapid initial diversification to infer unusual evolutionary processes. Analysis of discrete morphological characters shows that initial morphological diversification in the echinoderm subphylum Blastozoa was so pronounced that morphological diversity relative to taxonomic diversity was greatest in the Cambrian, whereas morphological diversity itself was greatest in the Middle and Upper Ordovician. Thus, a small number of Cambrian taxa sparsely occupied a large range in morphological space, whereas subsequent diversification involved expansion and filling of morphospace. A measure of clade-shape asymmetry and a method for statistical testing of clade shape are used to show that morphological diversity is significantly concentrated early in the history of the Blastozoa. The subphylum represents the highest biologic level at which temporal patterns of morphological diversity have been analyzed. Because this study is based on explicit morphological analysis, not taxonomic proxies for morphological diversity, the results are not artifacts of taxonomic practice.
Calcineurin mediates alpha-adrenergic stimulation of Na+,K(+)-ATPase activity in renal tubule cells.
Authors: Aperia A , Ibarra F , Svensson L B , Klee C , Greengard P
Published: May 2006

The alpha-adrenergic agonist oxymetazoline increased Na+,K(+)-ATPase activity of single proximal convoluted tubules dissected from rat kidney. Activation of the enzyme by oxymetazoline was prevented by either the alpha 1-adrenergic antagonist prazosin or the alpha 2-adrenergic antagonist yohimbine and was mimicked by the calcium ionophore A23187. The effect of oxymetazoline on Na+,K(+)-ATPase activity was prevented by a specific peptide inhibitor of calcineurin, as well as by FK 506, an immunosuppressant agent known to inhibit calcineurin; these results indicate that the action of oxymetazoline is mediated via activation of calcineurin (a calcium/calmodulin-dependent protein phosphatase). Activation of the Na+,K(+)-ATPase by either oxymetazoline or A23187 was associated with a greater than 2-fold increase in its affinity for Na+. The results provide a biochemical mechanism by which norepinephrine, released from renal nerve terminals, stimulates Na+ retention.
Identification of ubiquitous high-molecular-mass, heat-stable microtubule-associated proteins (MAPs) that are related to the Drosophila 205-kDa MAP but are not related to the mammalian MAP-4.
Authors: Kimble M , Khodjakov A L , Kuriyama R
Published: May 2006

AX3, a monoclonal antibody raised against isolated microtubule-organizing centers of Dictyostelium discoideum, stains microtubule-containing structures in species ranging from Dictyostelium to human. On immunoblots, the AX3 antibody recognizes heat-stable proteins in the 260- to 280-kDa molecular-mass range in a number of different species. The AX3 antigens from HeLa and embryonic mouse fibroblast cells coprecipitate with microtubules in vitro, indicating that these antigens are, indeed, MAPs. The AX3 antigens are not immunologically related to the mammalian MAP-2 or MAP-4 but are related to the 205-kDa MAP of Drosophila. This report describes a structural-type MAP in Dictyostelium and a MAP that is detected in a wide variety of species. The Drosophila 205-kDa MAP had previously been proposed to represent a member of the MAP-4 class of proteins. From the results reported here, however, it is suggested that proteins recognized by AX3 monoclonal antibody, including the Drosophila 205-kDa MAP, represent a distinct class of MAPs that has been widely conserved through evolution.
Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.
Authors: Jiang H P , Serrero G
Published: May 2006

We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA indicates that it encodes a protein of the 425 amino acids [tentatively named adipose differentiation-related protein (ADRP)] that does not have any similarity with sequences contained in the GenBank DNA and Protein Identification Resource protein data bases. Immunoblot of 1246 cell extracts with an antibody raised against the expressed ADRP shows that the 1246 cells contain a 50-kDa protein, the production of which increases as the cells differentiate. Localization of ADRP in 1246 cells indicates that ADRP is absent from nuclear and cytosolic fractions and is found as a membrane-associated protein. These results demonstrate that adipocyte differentiation is accompanied by early expression of a mRNA encoding a membrane-associated adipose differentiation related protein that is adipose tissue specific in vivo.
Identification of a transcriptional repressor down-regulated during preadipocyte differentiation.
Authors: Swick A G , Lane M D
Published: May 2006

During differentiation of 3T3-L1 preadipocytes into adipocytes, the transcription of adipocyte genes, including the stearoyl-CoA desaturase 2 (SCD2) gene, is activated. Transfection experiments with chimeric SCD2 promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs revealed a preadipocyte repressor element (PRE) capable of repressing transcription of the reporter gene in preadipocytes but not in adipocytes. DNase I protection and gel retardation analyses were used to localize the PRE site between nucleotides -435 and -410 of the SCD2 promoter and to identify a nuclear PRE binding protein present at high levels in preadipocytes and HeLa cells but lacking or inactive in adipocytes. Southwestern blot analysis indicated that the PRE binding protein has an apparent molecular mass of approximately 58 kDa. A single copy of the PRE site, inserted upstream of the simian virus 40 enhancer/promoter of pSV2CAT, was capable of strongly repressing transcription of the reporter gene in preadipocytes and HeLa cells but not in adipocytes. Taken together these results suggest that the PRE site and binding protein may regulate transcription of SCD2 and possibly other adipocyte genes by inhibiting their transcription in preadipocytes.
Regulated expression of the human acetylated low density lipoprotein receptor gene and isolation of promoter sequences.
Authors: Moulton K S , Wu H , Barnett J , Parthasarathy S , Glass C K
Published: May 2006

The acetylated low density lipoprotein (AcLDL) receptor is expressed on tissue macrophages after their differentiation from monocyte precursors and has been proposed to play a role in the generation of foam cells in atherosclerotic lesions. In the present studies, THP-1 human monocytic leukemia cells were used to investigate mechanisms responsible for expression of the AcLDL receptor gene after treatment with phorbol 12-myristate 13-acetate (TPA). TPA-dependent accumulation of AcLDL receptor mRNA was not detected until after a lag phase of 12 hr and was blocked by concurrent treatment with cycloheximide. In addition, the TPA-dependent induction of AcLDL receptor activity and mRNA levels was inhibited by retinoic acid and dexamethasone treatment. Isolation and sequence analysis of the promoter regions for the human and bovine AcLDL receptor genes indicated high sequence similarity. Binding sites for AP-1 proteins or other known transcription factors were not conserved between the two species, suggesting that novel factors are required for AcLDL receptor expression.
Dynamic changes in receptive-field size in cat primary visual cortex.
Authors: Pettet M W , Gilbert C D
Published: May 2006

Immediately after focal retinal lesions, receptive fields (RFs) in primary visual cortex expand considerably, even when the retinal damage is limited to the photoreceptor layer. The time course of these changes suggests that mere lack of stimulation in the vicinity of the RF accompanied by stimulation in the surrounding region causes the RF expansion. While recording from single cells in cat area 17, we simulated this pattern of stimulation with a pattern of moving lines in the visual field, masking out an area covering the RF of the recorded cell, thereby producing an "artificial scotoma." Over approximately 10 min this masking resulted in a 5-fold average expansion in RF area. Stimulating the RF center caused the field to collapse in size, returning to near its original extent; reconditioning with the masked stimulus led to RF reexpansion. Stimulation in the surrounding region was required for the RF expansion to occur--little expansion was seen during exposure to a blank screen. We propose that the expansion may account for visual illusions, such as perceptual fill-in of stabilized images and illusory contours and may constitute the prodrome of altered cortical topography after retinal lesions. These findings support the idea that even in adult animals RFs are dynamic, capable of being altered by the sensory context.
Analysis of dynamin isoforms in mammalian brain: dynamin-1 expression is spatially and temporally regulated during postnatal development.
Authors: Faire K , Trent F , Tepper J M , Bonder E M
Published: May 2006

In adult rat brain, the microtubule-associated protein dynamin is composed of a closely spaced polypeptide doublet of approximately 100 kDa. Using an antibody preparation that is monospecific for dynamin-1 (the higher molecular mass isoform) we examined the temporal and regional expression of dynamin-1 in developing rat brain. Analysis of whole rat brain homogenates established that prior to postnatal day 9, dynamin-1 was present only at very low levels and thereafter its expression steadily increased with adult levels being attained by postnatal day 23. In individual regions of the brain, dynamin-1 levels were highest in cortex, amygdala, and striatum, significantly lower in olfactory bulb, cerebellum, and midbrain, and lowest in brainstem. During postnatal development, each of the regions exhibited approximately the same time course of protein expression except for a slight lag in expression in olfactory bulb. The spatial and temporal patterns of expression of dynamin-1 correlate with the establishment and/or maintenance of mature neuronal structure and function rather than dendritic or axonal outgrowth.
Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei.
Authors: Li H , Roux S J
Published: May 2006

A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.
Genomic structure and chromosomal mapping of the mouse N-cadherin gene.
Authors: Miyatani S , Copeland N G , Gilbert D J , Jenkins N A , Takeichi M
Published: May 2006

N-cadherin is a member of the cadherin cell-cell adhesion receptor family that includes P-, E-, and R-cadherin and liver cell adhesion molecule (L-CAM). In this study, we determined the structure of the mouse N-cadherin gene by analyzing overlapping genomic clones obtained from a mouse genomic library. This gene consists of 16 exons that disperse over greater than 200 kilobases of genomic DNA. This large size of the N-cadherin gene, compared with its cDNA (4.3 kilobases), is ascribed to the fact that the first and second introns are 34.2 kilobases and greater than 100 kilobases long, respectively. When the N-cadherin gene was compared with that of L-CAM and P-cadherin, the exon-intron boundaries were found to be fully conserved between them, except that the P-cadherin first exon includes the first and second exons of the other two genes. Also, the second intron, which is equivalent to the first intron in P-cadherin, is exceptionally large and this structural feature is conserved in all of these genes. An interesting feature of the N-cadherin gene is that this gene has an extra 16th exon that is almost identical to the other exon, 100% in the coding region and 99% in the 3' untranslated region in the nucleotide level. We also determined the chromosomal localization of the N-cadherin gene by interspecific backcross analysis and found that this gene is localized in the proximal region of mouse chromosome 18. The E- and P-cadherin genes are tightly linked and located on chromosome 8 in this species. Thus, N-cadherin is unlinked to these other cadherin loci.
UV exposure reduces immunization rates and promotes tolerance to epicutaneous antigens in humans: relationship to dose, CD1a-DR+ epidermal macrophage induction, and Langerhans cell depletion.
Authors: Cooper K D , Oberhelman L , Hamilton T A , Baadsgaard O , Terhune M , LeVee G , Anderson T , Koren H
Published: May 2006

Increasing UVB radiation at the earth's surface might have adverse effects on in vivo immunologic responses in humans. We prospectively randomized subjects to test whether epicutaneous immunization is altered by prior administration of biologically equalized doses of UV radiation. Multiple doses of antigens on upper inner arm skin (UV protected) were used to elicit contact sensitivity responses, which were quantitated by measuring increases in skin thickness. If a dose of UVB sufficient to induce redness (erythemagenic) was administered to the immunization site prior to sensitization with dinitrochlorobenzene (DNCB), we noted a marked reduction in the degree of sensitization (P less than 0.0006) that was highly dose responsive (r = 0.98). Even suberythemagenic UV (less than a visible sunburn) resulted in a decreased frequency of strongly positive responses (32%) as compared to controls (73%) (P = 0.019). The rate of immunologic tolerance to DNCB (active suppression of a subsequent repeat immunization) in the groups that were initially sensitized on skin receiving erythemagenic doses of UV was 31% (P = 0.0003). In addition, a localized moderate sunburn appeared to modulate immunization with diphenylcyclopropenone through a distant, unirradiated site (41% weak responses) as compared to the control group (9%) (P = 0.05). Monitoring antigen presenting cell content in the epidermis revealed that erythemagenic regimens induced CD1a-DR+ macrophages and depleted Langerhans cells. In conclusion, relevant and even subclinical levels of UV exposure have significant down modulatory effects on the ability of humans to generate a T-cell-mediated response to antigens introduced through irradiated skin.
Protein isoforms encoded by the pX region of human T-cell leukemia/lymphotropic virus type I.
Authors: Koralnik I J , Gessain A , Klotman M E , Lo Monico A , Berneman Z N , Franchini G
Published: May 2006

The pX region of the human T-cell leukemia/lymphotropic virus type I (HTLV-I) contains at least four open reading frames (orfI-orfIV). orf III and orf IV encode the regulatory HTLV-I proteins Rex and Tax, which together modulate viral expression, and the p21rex protein of unknown function. By using the reverse transcriptase and polymerase chain reaction techniques on the RNA of an HTLV-I-infected cell culture, we uncovered the existence of alternatively spliced mRNAs generated through the use of three splice acceptor sites. These mRNAs encoded protein isoforms derived from the HTLV-I orf I (p12I) and orf II (p13II and p30II). An additional acceptor splice site, used in the processing of the env and tax/rex mRNAs and a singly spliced mRNA for the p21rex protein, was also identified. All of these HTLV-I mRNAs were also detected in freshly isolated cells from HTLV-I-infected individuals. Thus HTLV-I, like the human immunodeficiency virus type 1, has developed fine posttranscriptional mechanisms to increase the complexity of its genome.
Divergent evolution of pyrimidine biosynthesis between anaerobic and aerobic yeasts.
Authors: Nagy M , Lacroute F , Thomas D
Published: May 2006

A cDNA encoding the dihydroorotate dehydrogenase (DHOdehase; EC 1.3.3.1) of the yeast Schizosaccharomyces pombe was isolated by functional complementation in Saccharomyces cerevisiae. A divergent subcellular compartmentation of the DHOdehase of each yeast was shown. The DHOdehase from Sch. pombe was localized in the mitochondria whereas its homolog from S. cerevisiae was found to be cytosolic. The heterologous expression of the Sch. pombe enzyme in S. cerevisiae allowed us to demonstrate that the Sch. pombe DHOdehase activity requires the integrity of the mitochondrial electron transport chain. Indeed, the presence of a mutation inactivating cytochrome b abolished the complementation of a S. cerevisiae ura1 mutant by the corresponding Sch. pombe gene. By contrast, in vitro studies have revealed that the DHOdehase of S. cerevisiae uses fumarate as terminal electron acceptor. These results are discussed in relation to the anaerobic growth competence of the two yeasts and to the fermentative processes they use.
Accurate and rapid detection of heterozygous carriers of a deletion by combined polymerase chain reaction and high-performance liquid chromatography.
Authors: Asakawa J , Satoh C , Yamasaki Y , Chen S H
Published: May 2006

We have developed a technique to detect accurately heterozygous carriers of a deletion. Specific target sequences were amplified by the polymerase chain reaction (PCR), and the products subsequently were analyzed by high-performance liquid chromatography. Examples from four loci demonstrated that 24-27 cycles of amplification for a single-copy DNA, based on 50 ng of genomic DNA, results in excellent quantitation that readily permits the detection of heterozygous carriers of a deletion. We have demonstrated that triplex PCR (three targets in a single PCR) entails no loss of precision. We also have demonstrated that this method can accurately differentiate the heterozygous carriers of a deletion from normal individuals in four family studies, three for Duchenne muscular dystrophy patients and one for a hemophilia B patient.
Higher plant Ca(2+)-ATPase: primary structure and regulation of mRNA abundance by salt.
Authors: Wimmers L E , Ewing N N , Bennett A B
Published: May 2006

Calcium-dependent regulatory mechanisms participate in diverse developmentally, hormonally, and environmentally regulated processes, with the precise control of cytosolic Ca2+ concentration being critical to such mechanisms. In plant cells, P-type Ca(2+)-ATPases localized in the plasma membrane and the endoplasmic reticulum are thought to play a central role in regulating cytoplasmic Ca2+ concentrations. Ca(2+)-ATPase activity has been identified in isolated plant cell membranes, but the protein has not been characterized at the molecular level. We have isolated a partial-length cDNA (LCA1) and a complete genomic clone (gLCA13) encoding a putative endoplasmic reticulum-localized Ca(2+)-ATPase in tomato. The deduced amino acid sequence specifies a protein (Lycopersicon Ca(2+)-ATPase) of 1048 amino acids with a molecular mass of 116 kDa, eight probable transmembrane domains, and all of the highly conserved functional domains common to P-type cation-translocating ATPases. In addition, the protein shares approximately 50% amino acid sequence identify with animal sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases but less than 30% identity with other P-type ATPases. Genomic DNA blot hybridization analysis indicates that the Lycopersicon Ca(2+)-ATPase is encoded by a single gene. RNA blot hybridization analysis indicates the presence of three transcript sizes in root tissue and a single, much less abundant, transcript in leaves. Lycopersicon Ca(2+)-ATPase mRNA levels increase dramatically upon a 1-day exposure to 50 mM NaCl. Thus this report describes the primary structure of a higher-plant Ca(2+)-ATPase and the regulation of its mRNA abundance by salt stress.
Possible influences on the expression of X chromosome-linked dystrophin abnormalities by heterozygosity for autosomal recessive Fukuyama congenital muscular dystrophy.
Authors: Beggs A H , Neumann P E , Arahata K , Arikawa E , Nonaka I , Anderson M S , Kunkel L M
Published: May 2006

Abnormalities of dystrophin, a cytoskeletal protein of muscle and nerve, are generally considered specific for Duchenne and Becker muscular dystrophy. However, several patients have recently been identified with dystrophin deficiency who, before dystrophin testing, were considered to have Fukuyama congenital muscular dystrophy (FCMD) on the basis of clinical findings. Epidemiologic data suggest that only 1/3500 males with autosomal recessive FCMD should have abnormal dystrophin. To explain the observation of 3/23 FCMD males with abnormal dystrophin, we propose that dystrophin and the FCMD gene product interact and that the earlier onset and greater severity of these patients' phenotype (relative to Duchenne muscular dystrophy) are due to their being heterozygous for the FCMD mutation in addition to being hemizygous for Duchenne muscular dystrophy, a genotype that is predicted to occur in 1/175,000 Japanese males. This model may help explain the genetic basis for some of the clinical and pathological variability seen among patients with FCMD, and it has potential implications for understanding the inheritance of other autosomal recessive disorders in general. For example, sex ratios for rare autosomal recessive disorders caused by mutations in proteins that interact with X chromosome-linked gene products may display predictable deviation from 1:1.
Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors.
Authors: Lukat G S , McCleary W R , Stock A M , Stock J B
Published: May 2006

Bacterial motility and gene expression are controlled by a family of phosphorylated response regulators whose activities are modulated by an associated family of protein-histidine kinases. In chemotaxis there are two response regulators, CheY and CheB, that receive phosphoryl groups from the histidine kinase, CheA. Here we show that the response regulators catalyze their own phosphorylation in that both CheY and CheB can be phosphorylated in the complete absence of any auxiliary protein. Both CheY and CheB use the N-phosphoryl group in phosphoramidate (NH2PO3(2-)) as a phospho-donor. This enzymatic activity probably reflects the general ability of response regulators to accept phosphoryl groups from phosphohistidines in their associated kinases. It provides a general method for the study of activated response regulators in the absence of kinase proteins. CheY can also use intermediary metabolites such as acetyl phosphate and carbamoyl phosphate as phospho-donors. These reactions may provide a mechanism to modulate cell behavior in response to altered metabolic states.
Configurations of adsorbed hard spheres after diffusion in a gravitational field.
Authors: Senger B , Bafaluy F J , Schaaf P , Schmitt A , Voegel J
Published: May 2006

The deposition and adhesion of particles on a solid surface are governed by a great number of interplaying forces. In this paper we analyze, by means of computer simulations, the influence of (i) the short-range repulsive forces, modeled by hard sphere interactions, (ii) the gravitational forces, and (iii) the diffusion process of the particles in the fluid on the structure of the surface covered by the particles. In particular, the evolution of the limiting coverage, [symbol, see text] infinity (where [symbol, see text] is the reduced relative surface coverage), and the radial distribution, g(r), at the jamming limit, are determined as a function of the gravitational forces. These forces play an important role in many experiments performed on latex beads. Our results should stimulate new experiments in this field and, thus, be directly experimentally tested. It is shown, for example, that for polystyrene particles [symbol, see text] infinity is constant and equal to the random sequential adsorption jamming limit value for radii R not larger than 1 micron. It increases for 1 </= R </= 3 micron and tends, for higher R, to a plateau, whose value is approximately equal to 0.61. The tendency to a closer packing when R is large, and thus large gravitational forces, is confirmed by the shape of the radial distribution function. This phenomenon occurs not only for jammed surfaces but also for unsaturated surfaces.
Imaging of luciferase secretion from transformed Chinese hamster ovary cells.
Authors: Inouye S , Ohmiya Y , Toya Y , Tsuji F I
Published: May 2006

The blue luminescence characteristic of the marine ostracod crustacean Vargula hilgendorfii is from a simple, but highly specific, enzyme-substrate reaction. Light is emitted by the oxidation of Vargula luciferin (substrate) by molecular oxygen, a reaction catalyzed by luciferase. Stable transformants of Chinese hamster ovary cells carrying the Vargula luciferase gene secreted luciferase from discrete sites on the cell surface, and this secretion could be monitored in real time by the bioluminescence produced by the secreted luciferase in the presence of Vargula luciferin by using an image-intensifying technique. Addition of anti-Vargula luciferase IgG to the luminescing cells almost completely extinguished the luminescence, confirming that Vargula luciferase caused the luminescence.