bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic plasticity of Schwann cells and enteric glial cells in response to the microenvironment.
Authors: Dulac C , Le Douarin N M
Published: May 2006

We produced earlier a monoclonal antibody against Schwann cell myelin protein (SMP), a glycoprotein expressed on Schwann cells (SC) but not on satellite cells of the ganglia or enteric glial cells. We now studied whether SMP expression is environmentally regulated in the different compartments of the peripheral nervous system. Quail neural-crest cells from either mes-metencephalic, vagal, or truncal levels of the neuraxis were heterochronically associated with gut wall, skin, or muscle tissues from embryonic day (E) 7 to E11 chickens. Coculture of these chimeric organs revealed that as in normal development glial cells, characterized by HNK1 immunoreactivity and the quail nuclear marker, expressed the SMP phenotype exclusively in skin and muscle, failing to do so in gut. However, when SMP+ SC from quail sciatic nerves were cocultured with chicken gut, these cells rapidly lost their initial SMP immunoreactivity. In contrast, when associated with muscle and skin, SC remained SMP+, even in the complete absence of neuronal cells. Enteric plexuses from E8 to E15 quail gut express SMP+ and laminin when withdrawn from the intestinal-mesenchyme environment. These results show that SMP can be expressed by enteric glial cells and that the SC SMP phenotype is strongly inhibited by the gut-wall environment. Moreover, these results strongly suggest that these two types of glial cells belong to the same lineage and that their terminal phenotype is modulated through cell-to-cell interactions.
High density lipoprotein loses its effect to stimulate efflux of cholesterol from foam cells after oxidative modification.
Authors: Nagano Y , Arai H , Kita T
Published: May 2006

In this study, we performed oxidative modification of high density lipoprotein (HDL) in vitro. The amount of lipid peroxide increased when either HDL2 or HDL3 was incubated with phosphate-buffered saline containing 5 microM CuSO4 for 24 h at 37 degrees C, indicating that both fractions of HDL were oxidatively modified. This modification resulted in denaturation of apolipoprotein AI on SDS/PAGE and increased the negative charge on agarose gel electrophoresis. When incubated with macrophage-derived foam cells, native HDL caused a marked efflux of cholesterol from them, leading to a decrease in the amount of cholesteryl ester in the cells. However, oxidized HDL showed a lessened effect on the decrease of cholesteryl ester in foam cells. These data suggest that oxidative modification of HDL may stimulate development of atherosclerosis by limiting efflux of cholesterol from foam cells.
Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway.
Authors: Bartley G E , Viitanen P V , Pecker I , Chamovitz D , Hirschberg J , Scolnik P A
Published: May 2006

Carotenoids are orange, yellow, or red photo-protective pigments present in all plastids. The first carotenoid of the pathway is phytoene, a colorless compound that is converted into colored carotenoids through a series of desaturation reactions. Genes coding for carotenoid desaturases have been cloned from microbes but not from plants. We report the cloning of a cDNA for pds1, a soybean (Glycine max) gene that, based on a complementation assay using the photosynthetic bacterium Rhodobacter capsulatus, codes for an enzyme that catalyzes the two desaturation reactions that convert phytoene into zeta-carotene, a yellow carotenoid. The 2281-base-pair cDNA clone analyzed contains an open reading frame with the capacity to code for a 572-residue protein of predicted Mr 63,851. Alignment of the deduced Pds1 peptide sequence with the sequences of fungal and bacterial carotenoid desaturases revealed conservation of several amino acid residues, including a dinucleotide-binding motif that could mediate binding to FAD. The Pds1 protein is synthesized in vitro as a precursor that, upon import into isolated chloroplasts, is processed to a smaller mature form. Hybridization of the pds1 cDNA to genomic blots indicated that this gene is a member of a low-copy-number gene family. One of these loci was genetically mapped using restriction fragment length polymorphisms between Glycine max and Glycine soja. We conclude that pds1 is a nuclear gene encoding a phytoene desaturase enzyme that, as its microbial counterparts, contains sequence motifs characteristic of flavoproteins.
Rat liver canalicular membrane vesicles contain an ATP-dependent bile acid transport system.
Authors: Nishida T , Gatmaitan Z , Che M , Arias I M
Published: May 2006

The secretion of bile by the liver is primarily determined by the ability of the hepatocyte to transport bile acids into the bile canaliculus. A carrier-mediated process for the transport of taurocholate, the major bile acid in humans and rats, was previously demonstrated in canalicular membrane vesicles from rat liver. This process is driven by an outside-positive membrane potential that is, however, insufficient to explain the large bile acid concentration gradient between the hepatocyte and bile. In this study, we describe an ATP-dependent transport system for taurocholate in inside-out canalicular membrane vesicles from rat liver. The transport system is saturable, temperature-dependent, osmotically sensitive, specifically requires ATP, and does not function in sinusoidal membrane vesicles and right side-out canalicular membrane vesicles. Transport was inhibited by other bile acids but not by substrates for the previously demonstrated ATP-dependent canalicular transport systems for organic cations or nonbile acid organic anions. Defects in ATP-dependent canalicular transport of bile acids may contribute to reduced bile secretion (cholestasis) in various developmental, inheritable, and acquired disorders.
Analysis of pulsed-neutron powder diffraction patterns of the icosahedral quasicrystals Pd3Siu and AlCuLiMg (three alloys) as twinned cubic crystals with large units.
Authors: Pauling L
Published: May 2006

The low-Q peaks on three pulsed-neutron powder patterns (total, U differential, and Pd differential) of the icosahedral quasicrystal Pd3SiU have been indexed on the basis of an assumed cubic structure of the crystals that by icosahedral twinning form the quasicrystal. The primitive unit cube is found to have edge length 56.20 A and to contain approximately 12,100 atoms. Similar analyses of pulsed-neutron patterns of Al55Cu10Li35, Al55Cu10Li30Mg5, and Al510Cu125Li235Mg130 give values of the cube edge length 58.3, 58.5, and 58.4 A, respectively, with approximately 11,650 atoms in the unit cube. It is suggested that the unit contains eight complexes in the beta-W positions, plus some small interstitial groups of atoms, with each complex consisting of a centered icosahedron of 13 clusters, each of 116 atoms with the icosahedral structure found in the body-centered cubic crystal Mg32(Al,Zn)49.
Origin of a "bridge" intron in the gene for a two-domain globin.
Authors: Naito Y , Riggs C K , Vandergon T L , Riggs A F
Published: May 2006

Red cells of the clam Barbatia reeveana express two hemoglobins, one composed of 16- to 17-kDa chains and the other of 35-kDa chains. The nucleotide sequence of the cDNA encoding the 35-kDa chain shows that the polypeptide has two very similar heme-binding domains, which are joined without use of an additional bridging sequence. Two novel introns occur in the gene for the two-domain globin: one, the "precoding" intron, is located two bases 5' from the start codon, and the other, a "bridge" intron, separates the DNA sequences encoding the two domains. Close correspondence exists between the 3' end of the precoding intron and the 3' end of the bridge intron and between parts of the 3' noncoding region of the cDNA for the two-domain globin and the 5' end of the bridge intron. These observations indicate that the bridge intron arose by unequal crossing-over between two identical or very similar genes for a single-domain globin. This conclusion, together with the proposal that exons were initially independent "minigenes" [Gilbert, W. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, 901-905], suggests that many introns may have evolved from the 5' noncoding region of one gene and/or the 3' noncoding region of a second gene. This hypothesis implies that splice junctions would be associated with the original NH2 and COOH termini of proteins and provides an explanation for the observation that splice junctions usually map to protein surfaces. They do so because most NH2- and COOH-terminal residues are usually located on or near the surfaces of proteins.
Photoinduced degradation of the D1 polypeptide in isolated reaction centers of photosystem II: evidence for an autoproteolytic process triggered by the oxidizing side of the photosystem.
Authors: Shipton C A , Barber J
Published: May 2006

When the isolated D1/D2/cytochrome b559 complex was exposed to bright light, a distinctive pattern of D1 polypeptide fragments was observed under both aerobic and anaerobic conditions. The major degradation product had an apparent molecular mass of 24 kDa, while other fragments were detected at 17, 14, and 10 kDa by immunoblotting. This pattern was observed when the electron acceptors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone or silicomolybdate were present during illumination. It is known that these conditions stabilize P680+ chlorophyll and bring about the photooxidation and destruction of pigments in the reaction center, particularly chlorophyll absorbing at 670 nm and beta-carotene. When P680+ was not allowed to accumulate, either by omission of an electron acceptor or by addition of both an electron donor (Mn2+) and an acceptor, no breakdown fragments were observed. In the former case, however, some degradation of the D1 and D2 polypeptides did occur. Under conditions that gave rise to the characteristic D1 breakdown pattern, the D2 polypeptide was also degraded to specific fragments detected at about 29 and 21 kDa by immunoblotting. The results indicate that the photoinduced degradation of D1 (and D2) does not involve exogenous proteases but is most likely an autoproteolytic process. Moreover, our data indicate that the photochemical damage giving rise to D1 and D2 degradation occurs on the oxidizing rather than the reducing side of photosystem II and involves photooxidation of the accessory pigments. The results are discussed in terms of D1 and D2 turnover and photoinhibition.
Role of diversifying selection and gene conversion in evolution of major histocompatibility complex loci.
Authors: Ohta T
Published: May 2006

Genes at the major histocompatibility complex (MHC) in mammals are known to have exceptionally high polymorphism and linkage disequilibrium. In addition, these genes form highly complicated gene families that have evolved through gene conversion and unequal crossing-over. It has been shown recently that amino acid substitution at the antigen recognition site (ARS) is more rapid than synonymous substitution, suggesting some kind of positive natural selection working at the ARS. It is highly desirable to know the interactive effect of gene conversion and natural selection on the evolution and variation of MHC gene families. A population genetic model is constructed that incorporates both selection and gene conversion. Diversifying selection is assumed in which sequence diversity is enhanced not only between alleles at the same locus but also between duplicated genes. Expressed and nonexpressed loci are assumed as in the class I gene family of MHC, with gene conversion occurring among all loci. Extensive simulation studies reveal that very weak selection at individual amino acid sites in combination with gene conversion can explain the unusual pattern of evolution and polymorphisms. Here both gene conversion and natural selection contribute to enhancing polymorphism.
Loss of the endothelin signal pathway in C6 rat glioma cells persistently infected with measles virus.
Authors: Tas P W , Koschel K
Published: May 2006

Endothelin 1 causes a strong Ca2+ signal in C6 rat glioma cells as measured by fura-2 fluorescence. This endothelin 1-induced Ca2+ signal was not observed when the cells were persistently infected with a measles virus strain of subacute sclerosing panencephalitis (SSPE, strain Lec). Binding of 125I-labeled endothelin 1 to the C6/SSPE cells was less than 5% of the binding to the C6 control cells, suggesting that the impairment in signal transduction was due to a loss of binding sites for endothelin 1. Treatment of the C6/SSPE cells with measles antiserum resulted in the loss of expression of viral proteins located in the membrane as well as inside the cells (antigenic modulation), but it restored neither the endothelin 1-induced Ca2+ rise nor the 125I-endothelin 1 binding. Cocultivation of uninfected C6 cells with C6/SSPE cells (9:1 ratio) resulting in contact-mediated transmission of measles virus showed that the 125I-endothelin 1 binding activity was gradually lost as a consequence of persistent virus infection.
Coincidence cloning of Alu PCR products.
Authors: Aslanidis C , de Jong P J
Published: May 2006

Coincidence cloning allows the isolation of sequences held in common by two genomic DNA populations. Human DNA from two human-hamster hybrid cell lines was amplified by Alu-repeat primers (Alu PCR) and the products originating from the shared human chromosomal region were cloned. To achieve this, human sequences were amplified with very similar Alu primers from the two different human-hamster hybrid cell lines. The products were then digested with an appropriate restriction enzyme (either BamHI or Sal I), combined, denatured, and reannealed. The derived heteroduplex molecules (originating from the human regions common to both cell lines) had single BamHI and Sal I cohesive ends due to the primers used, so that they could be cloned in a double-digested plasmid vector. We used this method to enrich about 10-fold for Alu PCR products from the human chromosome 19q13.2 region, resulting in a region-specific clone collection. About 90% of the recombinants with BamHI-Sal I inserts are derived from the common region. This approach allows the boundaries for the regional probe isolation to be defined by combinations of hybrids rather than single hybrid cell lines, thus permitting greater flexibility in the selection of regions for probe isolation.
Free magnesium levels in normal human brain and brain tumors: 31P chemical-shift imaging measurements at 1.5 T.
Authors: Taylor J S , Vigneron D B , Murphy-Boesch J , Nelson S J , Kessler H B , Coia L , Curran W , Brown T R
Published: May 2006

We have studied a series of normal subjects and patients with brain tumors, by using 31P three-dimensional chemical shift imaging to obtain localized 31P spectra of the brain. A significant proportion of brain cytosolic ATP in normal brain is not complexed to Mg2+, as indicated by the chemical shift delta of the beta-P resonance of ATP. The ATP beta-P resonance position in brain thus is sensitive to changes in intracellular free Mg2+ concentration and in the proportion of ATP complexed with Mg because this shift lies on the rising portion of the delta vs. Mg2+ titration curve for ATP. We have measured the ATP beta-P shift and compared intracellular free Mg2+ concentration and fractions of free ATP for normal individuals (n = 6) and a limited series of patients with brain tumors (n = 5). In four of the five spectra obtained from brain tissue containing a substantial proportion of tumor, intracellular free Mg2+ was increased, and the fraction of free ATP was decreased, compared with normal brain.
Isolation and functional expression of a mammalian prohormone processing enzyme, murine prohormone convertase 1.
Authors: Korner J , Chun J , Harter D , Axel R
Published: May 2006

We have combined gene cloning with an assay for prohormone biosynthesis and processing in Xenopus oocytes to identify the genes that encode mammalian prohormone processing enzymes. The coinjection of RNA encoding murine prohormone convertase 1 (mPC1), a mammalian endoprotease, along with proopiomelanocortin RNA into an oocyte results in the appropriate cleavage after paired basic residues in the proopiomelanocortin polyprotein necessary to generate corticotropin. The ability of mPC1 to generate corticotropin, along with the observation that mPC1 is specifically expressed in endocrine and neuronal cells, suggests that the mPC1 gene encodes the endopeptidase responsible for the pathway of proopiomelanocortin cleavage observed in the anterior pituitary.
Clonality in myeloproliferative disorders: analysis by means of the polymerase chain reaction.
Authors: Gilliland D G , Blanchard K L , Levy J , Perrin S , Bunn H F
Published: May 2006

The myeloproliferative syndromes are acquired disorders of hematopoiesis that provide insights into the transition from somatic cell mutation to neoplasia. The clonal origin of specific blood cells can be assessed in patients with X chromosome-linked polymorphisms, taking advantage of random inactivation of the X chromosome. We have adapted the PCR for determination of clonality on as few as 100 cells, including individual colonies grown in culture. Amplifying a polymorphic portion of the X chromosome-linked phosphoglycerate kinase (PGK) gene after selective digestion of the active X chromosome with a methylation-sensitive restriction enzyme gave results fully concordant with standard Southern blotting of DNA samples from normal (polyclonal) polymorphonuclear cells (PMN) as well as clonal PMN from patients with myelodysplastic syndrome and polycythemia vera (PCV). We have used this technique to demonstrate heterogeneity of lineage involvement in patients with PCV. The same clinical phenotype may arise from clonal proliferation of different hematopoietic progenitors.
Spatial frequency tuning of single units in macaque supragranular striate cortex.
Authors: Born R T , Tootell R B
Published: May 2006

2-Deoxyglucose experiments have raised the possibility of a functional organization for spatial frequency in macaque striate cortex. To analyze this possibility with better spatial resolution, we made tangential microelectrode penetrations at constant eccentricity through supragranular striate cortex in 7 anesthetized, paralyzed macaque monkeys. We recorded from 121 single units. The data fell into two distinct populations with respect to mean preferred spatial frequency: (i) interblob cells (3.8 +/- 2.0 cycles/degree, n = 83) and (ii) blob cells (1.1 +/- 0.8 cycles/degree, n = 38; P less than 0.001). Beyond this, we found no evidence for an orderly mapping of spatial frequency optima. At blob-interblob borders, we observed abrupt shifts from low, relatively uniform spatial frequency optima (blobs) to higher optima that varied unsystematically (interblobs). The spatial frequency optima (low vs. high) and nature of the tuning curves (low-pass vs. band-pass) in blob vs. interblob cells correlate well with psychophysical measures of the same differences for the chrominance vs. luminance channels. These data are consistent with a functional subdivision of striate cortex in which blob cells carry information concerned predominantly with color and interblob neurons carry information important for form analysis.
Galactosyl ceramide or a derivative is an essential component of the neural receptor for human immunodeficiency virus type 1 envelope glycoprotein gp120.
Authors: Bhat S , Spitalnik S L , Gonzalez-Scarano F , Silberberg D H
Published: May 2006

This report demonstrates that galactosyl ceramide (GalCer) or a molecule derived from it may serve as an alternative receptor for human immunodeficiency virus in the nervous system. Recombinant gp120, an envelope glycoprotein of human immunodeficiency virus type 1, specifically binds to GalCer and its derivatives. This specificity was studied by inhibiting binding of radioiodinated gp120 to GalCer with antibodies to GalCer, antibodies to gp120, and an excess of unlabeled gp120. Binding activity was also removed by absorbing gp120 with liposomes containing GalCer. In addition, studies using natural and semisynthetic lipids indicate that the linkage between galactose and ceramide is essential for binding. The significance of an alternative receptor for human immunodeficiency virus in the nervous system is discussed.
Binding of the adenovirus VAI RNA to the interferon-induced 68-kDa protein kinase correlates with function.
Authors: Ghadge G D , Swaminathan S , Katze M G , Thimmapaya B
Published: May 2006

In human cells infected with adenovirus, the virus-associated RNA VAI blocks the activation of the interferon-induced double-stranded-RNA-dependent 68-kDa protein kinase (p68) and maintains normal levels of protein synthesis at late times after infection. VAI antagonizes the kinase activity by binding to p68. The structure of VAI consists of two long, base-paired stems connected by a complex short stem-loop structure. Previous work using a series of adenovirus mutants showed that the structural determinants of the VAI RNA that are essential for function reside in the central complex short stem-loop structure and adjacent base-paired regions (functional domain); the long duplex regions were found to be dispensable for function. To determine whether binding of VAI to p68 correlates with function and whether the structural determinants that are essential for function are also essential for binding, we studied the interaction of wild-type and several mutant VAI RNAs with p68 in whole cells. The p68-VAI complexes from mutant- and wild-type-infected cells were immunoprecipitated by an anti-p68 monoclonal antibody. The mutant RNAs that functioned efficiently in the cells bound to p68 efficiently in the cells, whereas functionally impaired mutants failed to bind to p68, indicating that the binding of the VAI RNA to p68 correlates well with function. In vitro binding assays with immunopurified p68 confirmed these observations. Secondary-structure analysis of several mutant VAI RNAs suggests that the binding does not depend on the long duplex regions but requires all the elements of the functional domain. We propose that the functional domain and the p68-binding domain of the VAI RNA are identical.
Morphology of a sensory neuron in Drosophila is abnormal in memory mutants and changes during aging.
Authors: Corfas G , Dudai Y
Published: May 2006

Several mutations in Drosophila impair learning and the cAMP cascade. We report here that the fine morphology of an identified mechanosensory neuron is abnormal in two of these mutants, dunce (dnc) and rutabaga (rut). The neuron innervating the antero-notopleural bristle was filled with horseradish peroxidase and studied at the light- and electron-microscopy level. In the mutants dnc and rut, this neuron has an abnormally large number of side branches and varicosities in a defined segment of the axon. In wild-type flies, age tends to decrease the number of side branches and variacosities in the same axonal segment that is affected by the mutations. Ultrastructural studies are compatible with the interpretation that the varicosities are potential synaptic sites. The results suggest that the cAMP cascade plays a role in shaping neuronal connectivity.
Localization of cholinergic differentiation factor/leukemia inhibitory factor mRNA in the rat brain and peripheral tissues.
Authors: Yamamori T
Published: May 2006

Sympathetic neurons display considerable plasticity in the neurotransmitter and neuropeptide phenotypes they express in vitro and in vivo. The cholinergic differentiation factor (CDF, also known as leukemia inhibitory factor, LIF) induces cultured rat sympathetic neurons to become cholinergic, without affecting their survival or growth. To understand the role of this factor in normal development, it is essential to determine where it is produced in situ. To localize CDF/LIF mRNA, a semiquantitative, reverse transcription-polymerase chain reaction method was employed. Actin and tubulin mRNA were used as internal controls, and two different sets of CDF/LIF primers were compared. In postnatal rat peripheral tissues, CDF/LIF mRNA was selectively localized in the target area of developing, sympathetic cholinergic neurons; the mRNA was not detected in the targets of sympathetic noradrenergic neurons. This finding supports the hypothesis that CDF/LIF is a target-derived neuronal differentiation factor. In postnatal rat brain, CDF/LIF mRNA is localized selectively in two parts of the visual system, visual cortex and superior colliculus. Thus, CDF/LIF may play a role in this system as well.
Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich "bubble" located within the rev-responsive region of viral mRNAs.
Authors: Heaphy S , Finch J T , Gait M J , Karn J , Singh M
Published: May 2006

The human immunodeficiency virus type 1 rev protein binds with high affinity (Kd less than 1-3 nM) to a purine-rich "bubble" containing bulged GG and GUA residues on either side of a double-helical RNA stem-loop located toward the 5' end of rev-response element RNA. High-affinity rev binding is maintained when the bubble is placed in heterologous stem-loop structures, but rev binding is reduced when either the bulged residues or flanking base pairs in the stem are altered. Rev binding to the purine-rich bubble nucleates assembly of long filamentous ribonucleoprotein structures containing polymers of rev bound to flanking RNA sequences. It is proposed that rev regulates human immunodeficiency virus RNA expression by selectively packaging viral transcripts carrying the rev-response element sequence into rod-like nucleoprotein complexes that block splicing of the packaged mRNAs.
Cell-specific expression of mRNAs encoding Na+,K(+)-ATPase alpha- and beta-subunit isoforms within the rat central nervous system.
Authors: Watts A G , Sanchez-Watts G , Emanuel J R , Levenson R
Published: May 2006

We have used in situ hybridization histochemistry to analyze the subcellular distribution of mRNAs encoding Na,K-ATPase alpha- and beta-subunit isoforms in the rat central nervous system. Substantial differences in the cell-specific pattern of expression were found for the genes encoding three isoforms of the alpha subunit. Transcripts of alpha 1-subunit gene were detected in virtually all cell types and structures examined. Expression of alpha 2-subunit mRNA was characteristic of glia, whereas alpha 3-subunit transcripts were predominant in neurons. Transcripts encoding the beta 1 subunit were detected in neurons, whereas beta 2-subunit mRNA expression was characteristic of glia. mRNA encoding both beta-subunit isoforms was present in choroidal epithelial cells. The distribution pattern of alpha- and beta-subunit mRNAs in structures throughout the central nervous system is consistent with the possibility of six structurally distinct Na+,K(+)-ATPase isoenzymes.
Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast.
Authors: Hamilton A J , Bouzayen M , Grierson D
Published: May 2006

The ethylene-forming enzyme (EFE), which catalyzes the last step in the biosynthesis of the plant hormone ethylene, has never been purified and no molecular probes are available. Recently, a putative cDNA clone for tomato EFE (pTOM13) has been identified by inhibiting ethylene synthesis with an antisense gene expressed in transgenic plants. A direct test of its function has been made by expression of a pTOM13 gene in Saccharomyces cerevisiae. After cloning artefacts were discovered in the 5' region of the cDNA, a corrected cDNA (pRC13) was created by the fusion of the 5' end of a genomic clone to the 3' end of the cDNA and expressed in S. cerevisiae. Cultures of transformed yeast converted 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, whereas control cells did not. This EFE activity displays similar characteristics to EFE found in plant tissue: it converts the trans isomer of the ACC analogue 1-amino-2-ethylcyclopropane-1-carboxylic acid to 1-butene in preference to the cis isomer, and it is strongly inhibited by cobaltous ions and 1,10-phenanthroline. Furthermore, information gained from the activity of effectors on yeast EFE activity supports the hypothesis that EFE is one of a group of hydroxylase enzymes.
Design and synthesis of a specific endothelin 1 antagonist: effects on pulmonary vasoconstriction.
Authors: Spinella M J , Malik A B , Everitt J , Andersen T T
Published: May 2006

The 21-amino acid vasoconstrictor peptide endothelin (Et) contains two disulfide bonds. We investigated the importance of the outer disulfide bond in Et-1 by replacing it with an amide linkage. Bioactivity was assessed in an isolated guinea pig lung preparation (perfused at constant flow with Ringer's solution/0.5% albumin) in which pulmonary artery pressure was monitored. Et-1 produced concentration-dependent pulmonary vasoconstriction at concentrations of 1 x 10(-10) M and higher. [Dpr1, Asp15]Et-1 (where Dpr is diaminopropionic acid), in which the outer disulfide was replaced by an amide bond and the inner disulfide was left intact, showed no agonist activity at 1 x 10(-6) M but 1 x 10(-7) M [Dpr1, Asp15]Et-1 inhibited Et-1-induced pulmonary vasoconstriction: effects of 1 x 10(-10) M 2 x 10(-10) M, and 1 x 10(-9) M Et-1 were inhibited by 98%, 75%, and 65%, respectively. Furthermore, this analog did not alter pulmonary vasoconstriction induced by thrombin, norepinephrine, or, most significantly, Et-3. A monocyclic Et-1 analog with the same sequence but in which the amide bond was not formed showed weak pulmonary vasoconstrictor activity (300-500 times less potent than Et-1) but had no antagonist activity. In addition, both the monocyclic control peptide and [Dpr1, Asp15]Et-1 competed effectively with 125I-labeled Et-1 for binding to cultured rat pulmonary artery smooth muscle cells. Thus, an Et-1 structural analog produced by replacement of the outer disulfide bond with an amide linkage displayed potent and specific Et-1 antagonism.
Staphylococcal exotoxins deliver activation signals to human T-cell clones via major histocompatibility complex class II molecules.
Authors: Spertini F , Spits H , Geha R S
Published: May 2006

We investigated whether staphylococcal exotoxins (SEs), in addition to their capacity to induce T-cell activation restricted by the T-cell receptor (TCR) beta-chain variable region, can deliver an activation signal to human T-cell clones through major histocompatibility complex (MHC) class II molecules. Eleven human T-cell clones (9 alpha beta TCR and 2 gamma delta TCR clones) of different antigenic specificities were tested for their capacity to proliferate in response to toxic shock syndrome toxin 1 (TSST-1) and two SEs, SEA and SEB. In the absence of accessory cells, only 4 alpha beta TCR clones were stimulated to proliferate, each by a single SE, and to mobilize intracellular free Ca2+ in response to that SE, events indicative of TCR engagement and, presumably, recognition restricted by the beta-chain variable region. In the presence of accessory cells, each of the 11 T-cell clones was stimulated to proliferate by any one of the three SEs tested. This apparently TCR-unrestricted SE-mediated polyclonal proliferation of T-cell clones occurred in the absence of an increase in intracellular free Ca2+ and was not dependent on the presence of MHC class II expression on accessory cells. In contrast, SE-mediated polyclonal proliferation did not occur in 3 alpha beta TCR clones derived from an MHC class II-deficient patient. Furthermore, all of the three SEs induced the proliferation of 4 natural-killer-cell clones, suggesting that expression of TCR/CD3 complex is not essential for SE-mediated polyclonal proliferation of activated lymphocytes. These results indicate that MHC class II molecules transduce activation signals to human T- and natural-killer-cell clones.
Pelizaeus-Merzbacher disease: a valine to phenylalanine point mutation in a putative extracellular loop of myelin proteolipid.
Authors: Pham-Dinh D , Popot J L , Boespflug-Tanguy O , Landrieu P , Deleuze J F , Boué J , Jollès P , Dautigny A
Published: May 2006

In the central nervous system, myelin proteolipid protein isoforms (PLP and DM20) play an essential structural role in myelination. It has been shown in several species that myelination is impaired by molecular defects resulting from single base mutations in the PLP gene. We have used DNA amplification by polymerase chain reaction to study the PLP gene coding regions from 17 patients in 15 unrelated families with similar Pelizaeus-Merzbacher phenotype. In one case amplification of peripheral nerve PLP/DM20 cDNAs revealed that a silent T----C transition was unrelated to the disease. In one family a nonsilent mutation was identified that leads to a phenylalanine substitution for valine-218 in PLP/DM20 proteins. We investigated the inheritance of the mutant allele in 19 subjects of this four-generation family and found a strict cosegregation of the Phe218 substitution with transmission and expression of the disease. The effect of the Val218----Phe mutation is discussed in the frame of a recently suggested topological model of PLP/DM20, according to which Val218 is part of an extracellular loop that connects the last two of four membrane-spanning alpha-helices.
Depolarization-induced changes in cellular energy production.
Authors: Erecińska M , Nelson D , Chance B
Published: May 2006

Addition of high concentrations of KC1 to preparations of rat brain synaptosomes incubated with either glucose or pyruvate caused a transient stimulation of oxygen uptake. This increased respiration was insensitive to 1 mM ouabain and 10 microM ruthenium red but was dependent upon the presence of calcium. With 40 mM KCl in the incubation medium, the levels of high-energy phosphate compounds in the synaptosomes were unaltered, whereas pyridine nucleotides underwent a rapid, albeit small and temporary, oxidation. It is postulated that there is a calcium-dependent mechanism in synaptosomes through which the function of the mitochondrial respiratory chain or of oxidative phosphorylation is stimulated directly without the involvement of either adenine nucleotides or mitochondrial dehydrogenases.
A human alcohol dehydrogenase gene (ADH6) encoding an additional class of isozyme.
Authors: Yasunami M , Chen C S , Yoshida A
Published: May 2006

The human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) gene family consists of five known loci (ADH1-ADH5), which have been mapped close together on chromosome 4 (4q21-25). ADH isozymes encoded by these genes are grouped in three distinct classes in terms of their enzymological properties. A moderate structural similarity is observed between the members of different classes. We isolated an additional member of the ADH gene family by means of cross-hybridization with the ADH2 (class I) cDNA probe. cDNA clones corresponding to this gene were derived from PCR-amplified libraries as well. The coding sequence of a 368-amino-acid-long open reading frame was interrupted by introns into eight exons and spanned approximately 17 kilobases on the genome. The gene contains a glucocorticoid response element at the 5' region. The transcript was detected in the stomach and liver. The deduced amino acid sequence of the open reading frame showed about 60% positional identity with known human ADHs. This extent of homology is comparable to interclass similarity in the human ADH family. Thus, the newly identified gene, which is designated ADH6, governs the synthesis of an enzyme that belongs to another class of ADHs presumably with a distinct physiological role.
Involvement of the interleukin 4 pathway in the generation of functional gamma delta T cells from human pro-T cells.
Authors: Bárcena A , Sánchez M J , de la Pompa J L , Toribio M L , Kroemer G , Martínez-A C
Published: May 2006

We have used the technique of in situ hybridization to investigate the transcription of genes encoding the CD3 complex and the lymphokine interleukin 4 (IL-4) by human pro-T cells--i.e., cells that phenotypically resemble those T-cell precursors that colonize the thymus during early intrathymic development. CD1-2-3-4-7+8-45+ pro-T cells isolated from postnatal thymi via immunoselection with a panel of specific monoclonal antibodies are already committed to the T-cell lineage because most of them transcribe the genes encoding the delta and epsilon chains of the CD3 complex. About half of such pro-T cells synthesize IL-4 mRNA in the absence of any exogenous stimulation. Upon culture with IL-4, pro-T cells extensively proliferate and differentiate into functionally competent, mature gamma delta T cells expressing a T-cell receptor repertoire similar to that of gamma delta T cells that can be found in postnatal thymus. The IL-4 response of pro-T cells is not mediated by induction of the interleukin 2 (IL-2)-IL-2 receptor pathway and, unlike IL-2-driven T-cell differentiation, does not require the presence of stromal cells. Taken altogether, these findings suggest that an autocrine IL-4-mediated pathway might be implicated in early thymocyte differentiation--namely, in the generation of T cells bearing the gamma delta T-cell receptor.
Site-directed mutations of Dictyostelium actin: disruption of a negative charge cluster at the N terminus.
Authors: Sutoh K , Ando M , Sutoh K , Toyoshima Y Y
Published: May 2006

Aspartic acid residues in the N-terminal negative charge cluster of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis of the actin gene. The mutant actins were expressed in Dictyostelium cells and were purified to homogeneity by HPLC. Functional properties of the mutant actins were compared with those of the wild-type actin. (i) In vitro assays of the sliding movement of actin filaments driven by myosin showed that the movement was slowed by the mutations. (ii) The mutations diminished the actin-activated ATPase activity of myosin in such a way that the maximum turnover rate at infinite actin concentration (Vmax) dropped sharply without an appreciable change in the apparent affinity of actin and myosin (Kapp). These results indicate that the N-terminal negative charge cluster of actin is essential for the ATP-dependent actin-myosin interaction.
Influence of site of expression on the fetal gamma delta T-cell receptor repertoire.
Authors: Kyes S , Pao W , Hayday A
Published: May 2006

The sequences of productive T-cell gamma/delta receptor transcripts were compared in different murine fetal tissues. Differences from tissue to tissue suggest that the sequence repertoires are at least in part the products of selection, presumably through interaction of T cells bearing the gamma delta receptor with fetal self-ligands.
T-cell-receptor-independent activation of cytolytic activity of cytotoxic T lymphocytes mediated through CD44 and gp90MEL-14.
Authors: Seth A , Gote L , Nagarkatti M , Nagarkatti P S
Published: May 2006

CD44 is a transmembrane glycoprotein found on a variety of cells including those of myeloid and lymphoid origin. CD44 is highly conserved among various species and is involved in the homing of lymphocytes and monocytes to lymph nodes, Peyer's patches, and sites of inflammation. In the present study, we demonstrate that monoclonal antibody (mAb) 9F3, directed against murine phagocytic glycoprotein 1 (CD44) expressed on cytotoxic T lymphocytes (CTLs), can trigger the lytic activity of CTLs and redirect CTL-mediated lysis to antigen-negative Fc receptor-positive target cells. Similar redirected lysis was also inducible using mAb MEL-14, directed against the lymphocyte homing receptor for endothelium (gp90MEL-14). The redirected lysis induced by mAbs 9F3 and MEL-14 was similar to that induced by mAbs against the alpha beta T-cell receptor or CD3. In contrast, mAbs directed against CD8, CD45R, and CD11a (LFA-1, lymphocyte function-associated antigen 1) failed to evoke lytic activity. The current study demonstrates that CD44 and gp90MEL-14 molecules, in addition to participating in T-cell homing and adhesion, may play a major role in delivering the transmembrane signal to the CTL that triggers the lytic activity, even when the T-cell receptor is not occupied. Such a mechanism may account for the nonspecific tissue damage seen at sites of CTL-mediated inflammation.
High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs.
Authors: Culp P , Nüsslein-Volhard C , Hopkins N
Published: May 2006

With the goal of developing techniques for DNA insertional mutagenesis in zebrafish, we established procedures for rapidly obtaining and injecting large numbers of fertilized eggs. Using either of two plasmid constructs, we injected uncut DNA into fertilized eggs at the one- or two-cell stage. Fish hatched from injected eggs were raised to sexual maturity, and the frequency of transgenic founder fish was determined by pair-mating the fish and testing DNA extracted from pools of their 16-hr-old offspring by the polymerase chain reaction (PCR) and then Southern analysis. Eggs injected with one of two different plasmids yielded no transgenic fish, but 7-25% (19 of 115 overall) of the eggs injected with the other plasmid transmitted the injected sequences to their offspring (F1). Of seven lines studied further, all were able to pass the foreign DNA sequences to the next (F2) generation. Inheritance in the F2 generation was Mendelian in the five lines tested. PCR and Southern analysis indicated that the plasmid sequences were present in multiple copies, probably tandemly arranged. Two founder fish carried more than one independent integration of the plasmid sequences. The line studied in more detail was a mosaic carrying two independently segregating copies of the transgene in one germ cell and a third copy in another germ-line precursor cell. The ability to obtain and inject large numbers of zebrafish eggs combined with a high frequency of germ-line integration may be steps toward the goal of being able to perform insertional mutagenesis with this organism.
Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures.
Authors: Langhoff E , Terwilliger E F , Bos H J , Kalland K H , Poznansky M C , Bacon O M , Haseltine W A
Published: May 2006

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells compose less than 1% of the circulating leukocytes and are nondividing cells. Highly purified preparations of dendritic cells were obtained using recent advances in cell fractionation. The results of these experiments show that dendritic cells, in contrast to monocytes and T cells, support the active replication of all strains of HIV-1 tested, including T-cell tropic and monocyte/macrophage tropic isolates. The dendritic cell cultures supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and adherent as well as nonadherent monocytes. Replication of HIV-1 in dendritic cells produces no noticeable cytopathic effect nor does it decrease total cell number. The ability of the nonreplicating dendritic cells to support high levels of replication of HIV-1 suggests that this antigen-presenting cell population, which is also capable of supporting clonal T-cell growth, may play a central role in HIV pathogenesis, serving as a source of continued infection of CD4+ T cells and as a reservoir of virus infection.
Glycosyl-phosphatidylinositol/inositol phosphoglycan: a signaling system for the low-affinity nerve growth factor receptor.
Authors: Represa J , Avila M A , Miner C , Giraldez F , Romero G , Clemente R , Mato J M , Varela-Nieto I
Published: May 2006

Nerve growth factor (NGF) exerts a variety of actions during embryonic development. At the early stages of inner ear development, NGF stimulates cell proliferation, an effect mediated through low-affinity receptors. We have studied the possibility that the glycosyl-phosphatidylinositol/inositol phosphoglycan (glycosyl-PtdIns/IPG) system is involved in transmitting this NGF signal. Endogenous glycosyl-PtdIns was characterized in extracts of cochleovestibular ganglia (CVGs) that incorporated [3H]glucosamine, [3H]galactose, [3H]myristic acid, and [3H]palmitic acid. Incubation of CVG with NGF produced a rapid and transient hydrolysis of glycosyl-PtdIns. Hydrolysis was complete at 100 ng/ml, and the half-maximal effect occurred at 25 ng/ml, overlapping with the concentration dependence of the mitogenic effect of NGF. An IPG was isolated from embryonic extracts. It had biological effects similar to those reported for the insulin-induced IPG in other tissues. It exerted a powerful mitogenic effect on CVG, comparable to that of NGF. Both the IPG- and NGF-induced cell proliferation were blocked by anti-IPG antibodies that recognized the endogenous IPG on a silica plate immunoassay. These results show that CVG possesses a fully active glycosyl-PtdIns/IPG signal transduction system and that the proliferative effects associated with NGF binding to low-affinity receptors require IPG generation.
Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA.
Authors: Stenger D C , Revington G N , Stevenson M C , Bisaro D M
Published: May 2006

Agrobacterium-mediated inoculation of Nicotiana benthamiana plants with Ti plasmids containing tandem genome repeats derived from different strains of the gemini-virus beet curly top virus (BCTV) resulted in the production of unit-length recombinant progeny genomes in systemically infected plants. When two putative plus-strand origins of replication were present in constructs used as inocula, a replicational escape mechanism was favored that resulted in progeny genomes of a single predominant genotype. The genotype was dependent upon the arrangement of repeated parental genomes in the inocula. Sequencing across the junction between parental BCTV strains in the recombinant progeny allowed mapping of the plus-strand origin of replication to a 20-base-pair sequence within the conserved hairpin found in all geminivirus genomes. In contrast, when inocula contained tandemly repeated BCTV genome sequences but only a single conserved hairpin, a number of different progeny genotypes were simultaneously replicated in infected plants, a result expected if unit-length viral genomes were generated by random intramolecular recombination events. These results and other considerations indicate that geminivirus DNA replication occurs by a rolling-circle mechanism.
Effects of injected Alzheimer beta-amyloid cores in rat brain.
Authors: Frautschy S A , Baird A , Cole G M
Published: May 2006

Although amyloid deposits have long been known to accumulate in Alzheimer disease (AD) brain, their origin and significance remain speculative. Because of the lack of an in vivo model where amyloid deposits can be induced, the relationship of the extracellular beta-amyloid deposits to other AD pathology has never been directly investigated. Therefore, we injected SDS-isolated amyloid cores into rat cortex and hippocampus. Similarly isolated lipofuscin fractions from control human brains were injected on the contralateral side. Rats were perfused and brains were examined immunohistochemically at 2 days, 7 days, and 1 month after injection. Alz-50, a monoclonal antibody against abnormally phosphorylated tau proteins, stained neurons along the cortical needle track at 2 but not 7 days after injection of either amyloid or lipofuscin. At 1 month, however, ubiquitin, Alz-50 antigen, and silver-positive structures were observed only in response to amyloid. In 7 of 10 animals, there was considerable neuronal loss in the hippocampal layers. In each instance, these effects were in the immediate vicinity of beta-protein immunoreactive material. Marked neuronal loss was never observed at any time after lipofuscin injection. These results indicate a neuronal response to amyloid. When preparations of mature plaque amyloid isolated from the AD brain are injected into the rat brain, they exert neurotoxic effects and induce antigens found in the AD brain.