bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective replication of simian immunodeficiency virus in a subset of CD4+ lymphocytes.
Authors: Gallatin W M , Gale M J , Hoffman P A , Willerford D M , Draves K E , Benveniste R E , Morton W R , Clark E A
Published: May 2006

Although all CD4+ cells theoretically are at risk for infection by human immunodeficiency viruses or the related simian immunodeficiency viruses found in Old World monkeys, only a small proportion of CD4+ lymphocytes from infected individuals have detectable virus. This suggests that immunodeficiency viruses may replicate predominantly in a minor subset or activated form of CD4+ T cells, a possibility we examined in macaques infected with a simian immunodeficiency virus isolate, SIV/Mne. Macaque CD4+ lymphocytes could be divided into two subtypes that differed in their level [high (hi) or low (lo)] of expression of a class of heterotypic adhesion receptors (HARs). In blood from animals infected with SIV/Mne, HARhi CD4+ T cells were lost selectively compared to HARlo CD4+ cells and, when cultured, exhibited 50-fold more recoverable reverse transcriptase activity. The HARhi CD4+ subset was also markedly more susceptible to productive infection following exposure to SIV/Mne in vitro. Both subsets are composed primarily of small resting lymphocytes. However, HARhi cells respond differentially to mitogenic stimulation and may thus be more likely to provide the cellular factors necessary to initiate or enhance virus replication. Thus, HAR expression may prove useful both as a prognostic indicator in immunodeficiency virus infection and as a tool to analyze pathogenesis of immunodeficiency viruses.
Saposin A: second cerebrosidase activator protein.
Authors: Morimoto S , Martin B M , Yamamoto Y , Kretz K A , O'Brien J S , Kishimoto Y
Published: May 2006

Saposin A, a heat-stable 16-kDa glycoprotein, was isolated from Gaucher disease spleen and purified to homogeneity. Chemical sequencing from its amino terminus and of peptides obtained by digestion with protease from Staphylococcus aureus strain V-8 demonstrated that saposin A is derived from proteolytic processing of domain 1 of its precursor protein, prosaposin. Processing of prosaposin (70 kDa) also generates three other previously reported saposin proteins, B, C, and D, from its second, third, and fourth domains. Similar to saposin C, saposin A stimulates the hydrolysis of 4-methylumbelliferyl beta-glucoside and glucocerebroside by beta-glucosylceramidase and of galactocerebroside by beta-galactosylceramidase, mainly by increasing the maximal velocity of both reactions. Saposin A is as active as saposin C in these reactions. Saposin A has no significant effect on other sphingolipid and 4-methylumbelliferyl glycoside hydrolases tested. Saposin A has two potential glycosylation sites that appear to be glycosylated. After deglycosylation, saposin A had a subunit molecular mass of 10 kDa and was as active as native saposin A. However, reduction and alkylation abolished the activation. A three-dimensional model comparing saposins A and C reveals significant sequence homology between them, especially preservation of conserved acidic and basic residues in their middle regions. Each appears to possess a conformationally rigid hydrophobic pocket stabilized by three internal disulfide bridges, with amphipathic helical regions interrupted by helix breakers.
Single potassium channels opened by opioids in rat locus ceruleus neurons.
Authors: Miyake M , Christie M J , North R A
Published: May 2006

Currents through single-ion channels were recorded in the cell-attached configuration from locus ceruleus neurons enzymatically dissociated from newborn rats. When the selective mu opioid receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol was in the patch-clamp electrode, unitary inward currents were observed with conductance of approximately 45 pS (measured at zero pipette potential, with 150 mM potassium in the recording electrode). Long silences, lasting many seconds to minutes, separated periods of activity of similar durations. Within such activity periods the distribution of closed times of the channels was best fitted by the sum of two exponential functions (time constants approximately 1 and 30 ms), and the durations of channel openings were fit by a single exponential function; mean open time increased from 2 to 120 ms as agonist concentration increased. Channel activity was not seen when high concentrations of opioids were applied to the neuron outside the patch-clamp recording electrode, indicating intimate coupling between receptor and potassium channel. Unitary currents with similar properties were also seen when pipettes contained alpha 2 adrenoceptor agonists or somatostatin. Taken with previous findings, the results indicate that mu opioid receptors, alpha 2 adrenoceptors, and somatostatin receptors can couple directly to membrane potassium channels through the local intermediary action of a GTP binding protein.
Temperature-derivative spectroscopy: a tool for protein dynamics.
Authors: Berendzen J , Braunstein D
Published: May 2006

A relaxation method that measures the derivative of a population with respect to temperature is introduced and used to study the recombination of CO to sperm whale myoglobin after a photolyzing flash. Measurement of the geminate process in the infrared CO-stretch bands shows distributed activation enthalpies with different distributions for each band, transitions between two bands that correspond to photolyzed ligands, and kinetic hole burning. The data are well described by gaussian enthalpy distributions; the results match and complement those of isothermal methods. The temperature-derivative technique is further used to explore the recombination of CO from outside the heme pocket.
Evidence for additional subunits associated to the mouse interleukin 2 receptor p55/p75 complex.
Authors: Saragovi H , Malek T R
Published: May 2006

Previous studies have indicated that high-affinity interleukin 2 receptors (IL-2R) are comprised of at least two distinct noncovalently associated subunits of Mr 55,000 (p55) and Mr 75,000 (p75). To biochemically characterize p75, we have directly isolated IL-2R using affinity precipitations with immobilized IL-2. We now report that at least some mouse p75 appears to exist as a disulfide-linked heterodimer with a subunit of Mr 22,000 (p22). These findings suggest that functional high-affinity mouse IL-2R may be comprised of at least three distinct subunits. The possibility of an even more complex structure is suggested by the coprecipitation of a protein of Mr 40,000-45,000 (p40) that may represent an additional IL-2R-associated protein.
Surface tension measurements show that chaotropic salting-in denaturants are not just water-structure breakers.
Authors: Breslow R , Guo T
Published: May 2006

Since the salting-in agents guanidinium chloride, urea, and lithium perchlorate increase the surface tension of water, the salting-in phenomenon does not reflect easier cavity formation in water. Therefore, these salting-in agents must be directly contributing to the solvation of a solute such as benzene in water, probably by a direct solvation interaction. The increased surface-tension effects do not overbalance these solvation effects since they are smaller than the large surface-tension increases with lithium chloride, a typical salting-out agent. The salting-in agent tetra-n-butylammonium chloride differs in that it lowers the surface tension of water. Thus, it probably contributes both to easier cavity formation and to direct solvation of the substrate. The previous findings that most salting-in agents switch to become salting-out agents in other polar solvents such as ethylene glycol and formamide but that tetra-n-butylammonium chloride does not switch in these solvents can be understood in terms of relative polarities.
Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis.
Authors: Menetski J P , Bear D G , Kowalczykowski S C
Published: May 2006

A question remaining to be answered about RecA protein function concerns the role of ATP hydrolysis during the DNA-strand-exchange reaction. In this paper we describe the formation of joint molecules in the absence of ATP hydrolysis, using adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) as nucleotide cofactor. Upon the addition of double-stranded DNA, the ATP[gamma S]-RecA protein-single-stranded DNA presynaptic complexes can form homologously paired molecules that are stable after deproteinization. Formation of these joint molecules requires both homology and a free homologous end, suggesting that they are plectonemic in nature. This reaction is very sensitive to magnesium ion concentration, with a maximum rate and extent observed at 4-5 mM magnesium acetate. Under these conditions, the average length of heteroduplex DNA within the joint molecules is 2.4-3.4 kilobase pairs. Thus, RecA protein can form extensive regions of heteroduplex DNA in the presence of ATP[gamma S], suggesting that homologous pairing and the exchange of the DNA molecules can occur without ATP hydrolysis. A model for the RecA protein-catalyzed DNA-strand-exchange reaction that incorporates these results and its relevance to the mechanisms of eukaryotic recombinases are presented.
A single clone of Staphylococcus aureus causes the majority of cases of toxic shock syndrome.
Authors: Musser J M , Schlievert P M , Chow A W , Ewan P , Kreiswirth B N , Rosdahl V T , Naidu A S , Witte W , Selander R K
Published: May 2006

Genetic relationships among 315 isolates of the bacterium Staphylococcus aureus expressing toxic shock syndrome toxin-1 (TSST-1) recovered primarily from humans with toxic shock syndrome (TSS) in five countries on two continents were determined by analyzing electrophoretically demonstrable allelic variation at 20 chromosomal enzyme loci. Forty-nine distinctive electrophoretic types (ETs), representing multilocus enzyme genotypes, were identified. Cluster analysis of the ETs revealed two major phylogenetic divisions separated at a genetic distance of 0.35 and seven branches diverging from one another at distances greater than or equal to 0.20. A single clone (ET 41) accounted for 88% of cases of TSS with a female urogenital focus and 53% of TSS cases involving nonurogenital (predominantly wound) infections. With few exceptions, strains representing different phylogenetic lines had characteristic TSST-1 gene (tst) restriction fragment length polymorphism patterns obtained by digestion of genomic DNA with Cla I. Strains recovered from ovine and bovine hosts with mastitis were genotypically distinct from the major human TSS clone. The expression of TSST-1 in cell lineages representing the total breadth of multilocus genotypic diversity in the species S. aureus as a whole is interpreted as evidence that the TSST-1 gene is evolutionarily old. The recovery of a single clone from the majority of individuals afflicted with TSS having a urogenital focus and from the genital tract of a large proportion of asymptomatic female carriers strongly suggests that this clone is especially well adapted for colonization of these anatomic sites.
Dopamine receptor gene expression by enkephalin neurons in rat forebrain.
Authors: Le Moine C , Normand E , Guitteny A F , Fouque B , Teoule R , Bloch B
Published: May 2006

In situ hybridization experiments were performed with brain sections from normal, control and haloperidol-treated rats to identify and map the cells expressing the D2 dopamine receptor gene. D2 receptor mRNA was detected with radioactive or biotinylated oligonucleotide probes. D2 receptor mRNA was present in glandular cells of the pituitary intermediate lobe and in neurons of the substantia nigra, ventral tegmental area, and forebrain, especially in caudate putamen, nucleus accumbens, olfactory tubercle, and piriform cortex. Hybridization with D2 and preproenkephalin A probes in adjacent sections, as well as combined hybridization with the two probes in the same sections, demonstrated that all detectable enkephalin neurons in the striatum contained the D2 receptor mRNA. Large neurons in caudate putamen, which were unlabeled with the preproenkephalin A probe and which may have been cholinergic, also expressed the D2 receptor gene. Haloperidol treatment (14 or 21 days) provoked an increase in mRNA content for D2 receptor and preproenkephalin A in the striatum. This suggests that the increase in D2 receptor number observed after haloperidol treatment is due to increased activity of the D2 gene. These results indicate that in the striatum, the enkephalin neurons are direct targets for dopamine liberated from mesostriatal neurons.
The allosteric glycine site of the N-methyl-D-aspartate receptor modulates GABAergic-mediated synaptic events in neonatal rat CA3 hippocampal neurons.
Authors: Gaiarsa J L , Corradetti R , Cherubini E , Ben-Ari Y
Published: May 2006

We report in this study that, in the presence of magnesium, bath application of micromolar concentrations of glycine have prominent effects on synaptic events and N-methyl-D-aspartate (NMDA) responses in neonatal but not in adult hippocampal slices. Intracellular recordings were made from 71 rat CA3 hippocampal neurons in neonatal slices. In keeping with our earlier study, during the first postnatal week, CA3 neurons exhibited giant depolarizing potentials (GDPs). These GDPs are mediated by gamma-aminobutyric acid (GABA) acting on type A GABA (GABAA) receptors and modulated presynaptically by NMDA receptors. In the majority of cells (18 out of 31), glycine (10-30 microM) increased the frequency of GDPs (from 0.14 to 0.29 Hz). This effect was mimicked by D-serine (10-20 microM) and blocked by the NMDA receptor antagonists D-(-)-2-amino-5-phosphonovalerate (50 microM) and DL-2-amino-7-phosphonoheptanoate (50 microM) and by the GABAA antagonist bicuculline (10 microM) but not by strychnine (1 microM). Subthreshold concentrations of glycine (or D-serine) and NMDA, when given together, enhanced synaptic noise and the frequency of GDPs. In the presence of tetrodotoxin (1 microM), glycine and D-serine (up to 50 microM) did not modify the NMDA-induced inward currents in CA3 pyramidal cells. However the reduction of NMDA-mediated currents by 7-chlorokynurenate (10-20 microM) was reversed by glycine and D-serine (100-200 microM). In contrast, glycine (up to 100 microM) had no effect on membrane potential, input resistance, or NMDA responses after postnatal day 10. It is concluded that GABA-mediated events are facilitated by glycine acting on presynaptically located NMDA receptors.
Manganese(II)-bicarbonate-mediated catalytic activity for hydrogen peroxide dismutation and amino acid oxidation: detection of free radical intermediates.
Authors: Yim M B , Berlett B S , Chock P B , Stadtman E R
Published: May 2006

To examine the structural identities of reactive free radicals and the mechanism of the oxidative modification of proteins, we used EPR and spin-trapping methods to investigate the oxidation of amino acids by H2O2 as well as the decomposition of H2O2 itself catalyzed by Mn(II) ions. Superoxide and hydroxyl radicals (O2-. and OH.) were trapped by a spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), in a reaction mixture containing Mn(II) and H2O2 in bicarbonate/CO2 buffer. When Hepes was used in place of bicarbonate buffer, superoxide radical was not observed, indicating the importance of bicarbonate buffer. With addition of L-leucine to a similar reaction mixture, a leucine-derived radical that replaced the DMPO-superoxide adduct was detected in the absence and presence of DMPO. Using various isotope-enriched L-leucines, we successfully identified this radical as a hydronitroxide, -OOC(R)CHNHO.. The data are consistent with the formation of a transient "caged" OH. in the inner coordination sphere of Mn(II). This caged OH. is likely to undergo an intramolecular hydrogen-atom abstraction from the Mn-bound H2O2 or amino acid. Two reaction schemes are proposed to account for the experimental results shown here and in the preceding papers.
Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY.
Authors: Bourret R B , Hess J F , Simon M I
Published: May 2006

The CheY protein is phosphorylated by CheA and dephosphorylated by CheZ as part of the chemotactic signal transduction pathway in Escherichia coli. Phosphorylation of CheY has been proposed to occur on an aspartate residue. Each of the eight aspartate residues of CheY was replaced by using site-directed mutagenesis. Substitutions at Asp-12, Asp-13, or Asp-57 resulted in loss of chemotaxis. Most of the mutant CheY proteins were still phosphorylated by CheA but exhibited modified biochemical properties, including reduced ability to accept phosphate from CheA, altered phosphate group stability, and/or resistance to CheZ-mediated dephosphorylation. The properties of CheY proteins bearing a substitution at position 57 were most aberrant, consistent with the hypothesis that Asp-57 is the normal site of acyl phosphate formation. Evidence for an alternate site of phosphorylation in the Asp-57 mutants is presented. Phosphorylated CheY is believed to cause tumbling behavior. However, a dominant mutant CheY protein that was not phosphorylated in vitro caused tumbling in vivo in the absence of CheA. This phenotype suggests that the role of phosphorylation in the wild-type CheY protein is to stabilize a transient conformational change that can generate tumbling behavior.
Labile disulfide bonds in human placental insulin receptor.
Authors: Finn F M , Ridge K D , Hofmann K
Published: May 2006

The disulfide crosslinking pattern of human placental insulin receptor was investigated using selective reduction with tributylphosphine followed by alkylation with N-[3H]ethylmaleimide. Insulin receptor contains a single sulfhydryl group in each beta subunit whose alkylation with N-[3H]ethylmaleimide inhibits receptor autophosphorylation. Alkylation is partially inhibited by ATP or the nonhydrolyzable substrate analog adenosine 5'-[beta,gamma-imido]triphosphate when the nucleotides are added as Mn2+ complexes. Neither insulin nor 6 M guanidinium chloride renders additional sulfhydryl groups accessible to alkylation. When the receptor is reduced under drastic conditions with tributylphosphine in guanidinium chloride, 32 of the 37 sulfhydryl groups in the receptor's alpha subunit can be alkylated with N-[3H]ethylmaleimide. Surprisingly only three of the 10 cysteines in the beta subunit become titratable under identical conditions. By using highly selective reducing conditions, we were able to determine quantitatively the maximum number of disulfide bridges that link the two alpha beta halves to form the tetrameric structure and those that couple the alpha to the beta subunits. Liberation of two sulfhydryl groups in the alpha and one in the beta subunit resulted in formation of alpha beta dimers. Free beta subunit was formed when an additional disulfide bond was reduced. It is remarkable that the tetrameric structure of this highly complex receptor molecule, which contains a large number of cysteine residues, is maintained by such a small number of disulfide bonds. Three models of the arrangement of the labile disulfide bonds, consistent with these findings, are proposed.
A simple model for the immune network.
Authors: Parisi G
Published: May 2006

In this note I present a simple model for the idiotypic network among antibodies and study its relevance for the maintenance of immunological memory; in particular, the memory capacity of such a model is studied. Some of the similarities with the spin glass model and with neural networks are discussed.
Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells.
Authors: Wilson J M , Danos O , Grossman M , Raulet D H , Mulligan R C
Published: May 2006

Recombinant retroviruses encoding human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans.
Interleukin 5 is required for the blood and tissue eosinophilia but not granuloma formation induced by infection with Schistosoma mansoni.
Authors: Sher A , Coffman R L , Hieny S , Scott P , Cheever A W
Published: May 2006

Eosinophils are thought to play a major role in the immunobiology of schistosomiasis. To investigate the immunologic basis of the eosinophil response and directly assess the function of eosinophils in egg-induced pathology, mice infected with Schistosoma mansoni were injected with a monoclonal antibody produced against interleukin 5 (IL-5), a cytokine previously shown to stimulate eosinophil differentiation in vitro. This treatment suppressed the generation of eosinophil myelocyte precursors in the bone marrow and reduced to background levels the numbers of mature eosinophils in the marrow, in circulation, and within acute schistosome egg granulomas. Nevertheless, granulomas in the anti-IL-5-treated/eosinophil-depleted mice at 8 weeks of infection were only marginally smaller than those in animals injected with control monoclonal antibody, and hepatic fibrosis was comparable in the two groups. Additional parameters such as worm burden, egg output, and serum IgE levels were unaltered by the anti-IL-5 treatment. In contrast, infected animals injected with monoclonal antibody against gamma interferon (IFN-gamma) displayed circulating eosinophil levels that were elevated with respect to control mice, possibly because of an enhanced release of mature eosinophils from the marrow, and developed egg granulomas that were indistinguishable in size and cellular composition from those in control animals. Immunologic assays revealed that lymphocytes from acutely infected mice produce large quantities of IL-5 but minimal IFN-gamma when stimulated with either egg antigen or mitogen. Taken together, these results indicate that neither IL-5 nor eosinophils are essential for egg-induced pathology but suggest that lymphocytes that belong to the IL-5-producing TH2 subset predominate during acute infection and may induce granuloma formation by the production of other cytokines.
2-Azido-[32P]NAD+, a photoactivatable probe for G-protein structure: evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of alpha interacts with both alpha and gamma subunits.
Authors: Vaillancourt R R , Dhanasekaran N , Johnson G L , Ruoho A E
Published: May 2006

A radioactive and photoactivatable derivative of NAD+, 2-azido-[adenylate-32P]NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the alpha subunit (alpha T) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of alpha T followed by light activation of the azide moiety of 2-azido-[adenylate-32P]ADP-ribose produced four crosslinked species involving the alpha and gamma subunits of the GT heterotrimer: an alpha trimer (alpha-alpha-alpha), and alpha-alpha-gamma crosslink, an alpha dimer (alpha-alpha), and an alpha-gamma crosslink. The alpha trimer, alpha-alpha-gamma complex, alpha dimer, and alpha-gamma complexes were immunoreactive with alpha T antibodies. The alpha-alpha-gamma and the alpha-gamma complexes were immunoreactive with antisera recognizing gamma subunits. No evidence was found for crosslinking of alpha T to beta T subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-[adenylate-32P]ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of alpha T in the crosslinked oligomers to alpha monomers, indicative of intermolecular photocrosslinking, and to gamma monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the alpha T-carboxyl terminus, is oriented toward and in close proximity to the gamma subunit.
Repopulation of the atrophied thymus in diabetic rats by insulin-like growth factor I.
Authors: Binz K , Joller P , Froesch P , Binz H , Zapf J , Froesch E R
Published: May 2006

Atrophy of the thymus is one of the consequences of severe insulin deficiency. We describe here that the weight and the architecture of the thymus of diabetic rats is restored towards normal not only by insulin but also by insulin-like growth factor I (IGF-I) treatment. In contrast to insulin, this effect of IGF-I occurs despite persisting hyperglycemia and adrenal hyperplasia. We also investigated the in vivo effect of IGF-I on replication and differentiation of thymocytes from streptozotocin-induced diabetic rats. Thymocytes from diabetic rats incorporated less [3H]thymidine than did thymocytes from healthy rats. Insulin, as well as IGF-I treatment of diabetic rats increased [3H]thymidine incorporation by thymocytes. Flow cytometry of thymocytes labeled with monoclonal antibodies revealed a decreased expression of the Thy-1 antigen in diabetic rats compared with control rats. In addition, a major deficiency of thymocytes expressing simultaneously the W3/25 and the Ox8 antigens (corresponding to immature human CD4+/CD8+ thymocytes) was observed. These changes were restored towards normal by insulin as well as by IGF-I treatment. The antibody response to a T cell-dependent antigen (bovine serum albumin) was comparable in normal and diabetic rats. We conclude that IGF-I has important effects on the thymocyte number and the presence of CD4+/CD8+ immature cells in the thymus of diabetic rats despite persisting hyperglycemia. However, helper T-cell function for antibody production appears to be preserved even in the severely diabetic state.
Refinement of protein dynamic structure: normal mode refinement.
Authors: Kidera A , Go N
Published: May 2006

An x-ray crystallographic refinement method, referred to as the normal mode refinement, is proposed. The Debye-Waller factor is expanded in terms of the effective normal modes whose amplitudes and eigenvectors are experimentally determined by the crystallographic refinement. In contrast to the conventional method, the atomic motions are treated generally as anisotropic and concerted. This method is assessed by using the simulated x-ray data given by a Monte Carlo simulation of human lysozyme. In this article, we refine the dynamic structure by fixing the average static structure to exact coordinates. It is found that the normal mode refinement, using a smaller number of variables, gives a better R factor and more information on the dynamics (anisotropy and collectivity in the motion).
Regulation of alternative splicing in the generation of isoforms of the mouse Ly-5 (CD45) glycoprotein.
Authors: Saga Y , Lee J S , Saraiya C , Boyse E A
Published: May 2006

Alternative splicing generates various Ly-5 glycoprotein isoforms of the cell surface that typify different cell lineages and stages of hematopoietic differentiation in the mouse; exons 4-6 are incorporated to generate a B-cell isoform (B220) and excluded from a T-cell isoform (T200), the other coding exons (3 and 7-33) being shared. As a first step to understanding the mechanisms regulating Ly-5 alternative splicing, and thus determining Ly-5 isoforms, a minigene representing exons 3-7 was transfected into Ly-5-expressor T cells and B cells and into nonexpressor L cells for comparison of splicing patterns. We conclude that all the information required for faithful splice-site selection according to cell type is contained within the resulting pre-mRNA. The splicing pattern manifested by nonexpressor L cells may represent a default and nonregulated type. We postulate trans-acting factor(s) to account for the selection of appropriate exons, and we provide support for this interpretation from analysis of fused hybrid T-B cells, which exhibited B-cell specific Ly-5 transcripts. Splicing patterns were well conserved despite substantial disruption of constructs. However, extensive deletion analyses suggested that cis sequences flanking and within exon 6 affect the exclusion of that exon in T cells.
Effect of controlled adventitial heparin delivery on smooth muscle cell proliferation following endothelial injury.
Authors: Edelman E R , Adams D H , Karnovsky M J
Published: May 2006

Continuous intravenous infusion of heparin suppresses smooth muscle cell proliferation in rats after endothelial injury but may lead to hemorrhage and other complications. The anticoagulant property has been removed from chemically modified heparin without loss of antiproliferative effect but use of such compounds is still limited. In this study ethylene-vinyl acetate copolymer matrices containing standard and modified heparin were placed adjacent to rat carotid arteries at the time of balloon dendothelialization. After 14 days arterial occlusion by smooth muscle cell proliferation was defined. Matrix delivery of both heparin compounds effectively diminished this proliferation in comparison to controls without producing systemic anticoagulation or side effects. In addition, this mode of therapy appeared more effective than the administration of the same agents by either intravenous pumps or heparin/polymer matrices placed in a subcutaneous site distant from the injured carotid artery. Thus, heparin's inhibition of smooth muscle cell proliferation after vascular injury might be most effective within the microenvironment of the injured vessel wall, and the accelerated atherosclerosis or restenosis that often follows angioplasty and other vascular interventions might best be treated with site-specific therapy.
Protein synthesis during acquisition of long-term facilitation is needed for the persistent loss of regulatory subunits of the Aplysia cAMP-dependent protein kinase.
Authors: Bergold P J , Sweatt J D , Winicov I , Weiss K R , Kandel E R , Schwartz J H
Published: May 2006

Depending on the number or the length of exposure, application of serotonin can produce either short-term or long-term presynaptic facilitation of Aplysia sensory-to-motor synapses. The cAMP-dependent protein kinase, a heterodimer of two regulatory and two catalytic subunits, has been shown to become stably activated only during long-term facilitation. Both acquisition of long-term facilitation and persistent activation of the kinase is blocked by anisomycin, an effective, reversible, and specific inhibitor of protein synthesis in Aplysia. We report here that 2-hr exposure of pleural sensory cells to serotonin lowers the concentration of regulatory subunits but does not change the concentration of catalytic subunits, as assayed 24 hr later; 5-min exposure to serotonin has no effect on either type of subunit. Increasing intracellular cAMP with a permeable analog of cAMP together with the phosphodiesterase inhibitor isobutyl methylxanthine also decreased regulatory subunits, suggesting that cAMP is the second messenger mediating serotonin action. Anisomycin blocked the loss of regulatory subunits only when applied with serotonin; application after the 2-hr treatment with serotonin had no effect. In the Aplysia accessory radula contractor muscle, prolonged exposure to serotonin or to the peptide transmitter small cardioactive peptide B, both of which produce large increases in intracellular cAMP, does not decrease regulatory subunits. This mechanism of regulating the cAMP-dependent protein kinase therefore may be specific to the nervous system. We conclude that during long-term facilitation, new protein is synthesized in response to the facilitatory stimulus, which changes the ratio of subunits of the cAMP-dependent protein kinase. This alteration in ratio could persistently activate the kinase and produce the persistent phosphorylation seen in long-term facilitated sensory cells.
Transient calcium release induced by successive increments of inositol 1,4,5-trisphosphate.
Authors: Meyer T , Stryer L
Published: May 2006

Many hormonal, neurotransmitter, and sensory stimuli trigger the formation of inositol 1,4,5-trisphosphate, which in turn releases calcium from intracellular stores. We report here that inositol 1,4,5-trisphosphate-induced calcium release from saponin-permeabilized rat basophilic leukemia cells at 37 degrees C is markedly biphasic, in contrast with nearly monophasic release kinetics at 11 degrees C. Hepatoma, PC-12 neuronal cells, and several other cell types exhibit similar biphasic release at 37 degrees C. The biphasic kinetics are not due to degradation of inositol 1,4,5-trisphosphate or to increased Ca2(+)-ATPase pump activity. Biphasic calcium release was also seen when ATP was quenched to less than 0.4 microM by adding hexokinase and glucose, suggesting that phosphorylation is not involved. External calcium (100 nM-600 nM) range had little influence on the biphasic kinetics. Rapid-mixing experiments revealed that rapid efflux of calcium is followed in approximately 0.5 s by a 30-fold slower efflux. Most striking, successive additions of the same amount of inositol 1,4,5-trisphosphate induced short bursts of calcium release of similar size. This retention of responsiveness, which we term increment detection, may be a distinct mode of signal transduction. Like inactivation and adaptation, increment detection gives rise to transient responses to sustained stimuli. Systems exhibiting inactivation, adaptation, and increment detection differ in their responsiveness (none, partial, and full, respectively) to stepwise increases in stimulus intensity. Increment detection could be advantageous in generating receptor-triggered calcium oscillations.
Sequence analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit.
Authors: Bozak K R , Yu H , Sirevåg R , Christoffersen R E
Published: May 2006

The ripening of avocado fruit is associated with the expression of a number of mRNAs concomitant with overt changes in texture and flavor. Two overlapping cDNAs for a mRNA that accumulates during ripening were identified. Sequence analysis of these two cDNAs revealed a polypeptide of 471 amino acids with characteristics of a typical P-450: an N-terminal hydrophobic membrane anchor, a conserved heme-binding domain in the C-terminal region, and patches of similarity to various P-450 family members. Further evidence that this polypeptide represents a cytochrome P-450 oxidase comes from the recent isolation and characterization of a cytochrome P-450 from ripe avocado mesocarp [O'Keefe, D. P. & Leto, K. J. (1989) Plant Physiol. 89, 1141-1149]. The N terminus of the predicted polypeptide in the cDNAs is identical to the N terminus of the purified avocado P-450. Gel blot analysis of RNA from fruit at various stages of ripening showed the accumulation of an 1800-nucleotide P-450 mRNA that hybridized to the P-450 cDNA. The P-450 protein predicted by the avocado cDNA sequence shares less than 40% positional identity with any known P-450 gene family. We propose therefore that it be placed in a separate family, P450LXXI, and that the corresponding gene from avocado be named cyp71A1.
Human immunodeficiency virus causes mononuclear phagocyte dysfunction.
Authors: Baldwin G C , Fleischmann J , Chung Y , Koyanagi Y , Chen I S , Golde D W
Published: May 2006

There is compelling clinical evidence for dysfunction of the mononuclear phagocyte system in patients with AIDS, which is believed due in part to loss of T-cell cooperativity. The direct consequences of human immunodeficiency virus infection on macrophage function are unknown. To address this question we infected normal human macrophages in vitro with a monocytotropic strain of human immunodeficiency virus and performed assays to quantify their extra- and intracellular killing ability. Human immunodeficiency virus-infected macrophages were significantly less effective than control cells in mediating antibody-dependent cell-mediated cytotoxicity against leukemic cell targets and intracellular killing of Candida pseudotropicalis. The functional defects were profound, related temporarily to active virus production by the macrophages, and could not be overcome by granulocyte-macrophage colony-stimulating factor. Treatment of macrophages with 3'-azido-3'-deoxythymidine (AZT) 6 days after infection caused a marked decrease in virus production and prevented development of the intracellular killing functional defect. The results suggest that early antiviral therapy may be useful in preventing or mitigating some virus-induced mononuclear phagocyte dysfunction.
Topographical relationship between beta-amyloid and tau protein epitopes in tangle-bearing cells in Alzheimer disease.
Authors: Spillantini M G , Goedert M , Jakes R , Klug A
Published: May 2006

Double-labeling immunohistochemistry was used to investigate the topographical relationship between beta-amyloid and tau protein epitopes present in cells bearing neurofibrillary tangles found in the hippocampal formation of patients with Alzheimer disease. An antiserum raised against the amino terminus of beta-amyloid stained numerous tangle-bearing cells and other bodies ("extracellular tangles"), but double labeling showed that the beta-amyloid staining is invariably peripheral to that of the tau-positive tangle proper. This and other results suggest that the extracellular amyloid plaques and the intracellular neurofibrillary tangles are biochemically distinct but may result from related pathological events that originate at the level of the nerve cell and lead to its degeneration.
Continuous monitoring of receptor-mediated changes in the metabolic rates of living cells.
Authors: Owicki J C , Parce J W , Kercso K M , Sigal G B , Muir V C , Venter J C , Fraser C M , McConnell H M
Published: May 2006

Activation of beta-adrenergic or muscarinic acetylcholine receptors expressed in transfected cells or epidermal growth factor receptors in human keratinocytes produces 15% to 200% changes in cellular metabolic rates. Changes in cell metabolism were monitored continuously with a previously described silicon-based microphysiometer that detects small changes in extracellular pH. The amplitude and kinetics of the metabolic changes depend upon several factors including pretreatment of the cells prior to receptor stimulation, the dose of hormone/neurotransmitter used, and the receptor complement of the cells. Responses are receptor specific; cells transfected with receptor genes respond only to the appropriate hormone/transmitter, whereas control (nontransfected) cells or cells transfected with different receptors exhibit no response. The specificity of the responses was further documented by using pharmacological antagonists. In Chinese hamster ovary (CHO) cells transfected with human beta 2-adrenergic receptors, isoproterenol produces a 20-60% increase in the rate of extracellular acidification with an EC50 of 4 nM, a response that is competitively antagonized by (-)-propranolol. The EC50 for the isoproterenol response is shifted from 4 nM to 100 nM in the presence of 3 nM (-)-propranolol. The kinetics of the metabolic response induced by beta-adrenergic receptor stimulation are markedly slower than those elicited by muscarinic receptor agonists. The maximal metabolic response in cells transfected with beta-adrenergic receptors peaks at approximately 12 min as compared with less than 30 sec in cells transfected with muscarinic receptors, perhaps reflecting activation of different second-messenger pathways. These findings illustrate an alternative means of studying cellular responses to hormones and neurotransmitters and suggest that metabolic changes will be generally useful for detecting the consequences of receptor-ligand interactions.
Immunization of Aotus monkeys with Plasmodium falciparum blood-stage recombinant proteins.
Authors: Herrera S , Herrera M A , Perlaza B L , Burki Y , Caspers P , Döbeli H , Rotmann D , Certa U
Published: May 2006

The current spread of multidrug-resistant malaria demands rapid vaccine development against the major pathogen Plasmodium falciparum. The high quantities of protein required for a worldwide vaccination campaign select recombinant DNA technology as a practical approach for large-scale antigen production. We describe the vaccination of Aotus monkeys with two recombinant blood-stage antigens (recombinant p41 and 190N) that were considered as vaccine candidates because parasite-derived antigen preparations could protect susceptible monkeys from an otherwise lethal malaria infection. In contrast to the natural antigen, recombinant p41 protein (P. falciparum aldolase) could not protect monkeys, although all animals seroconverted. 190N antigen, a recombinant protein containing conserved sequences of the major merozoite surface antigen p190, protected two of five monkeys from critical levels of infection with the highly virulent FVO isolate of P. falciparum. However, the B- and T-cell responses to 190N antigen were similar in protected and unprotected animals so that other unknown factors may contribute to protection. Higher purity or lack of protective epitopes or different structure of protective epitopes in the recombinant proteins might explain the better performance of parasite-derived antigens in vaccination trials. The partial protection obtained with 190N antigen suggests that this molecule could contribute to a vaccine mixture against P. falciparum.
Augmentation of glutathione in the fluid lining the epithelium of the lower respiratory tract by directly administering glutathione aerosol.
Authors: Buhl R , Vogelmeier C , Critenden M , Hubbard R C , Hoyt R F , Wilson E M , Cantin A M , Crystal R G
Published: May 2006

Glutathione (GSH), a cysteine-containing tripeptide, functions as an antioxidant, provides cells with cysteine, and is required for optimal function of the immune system. Because the epithelial-lining fluid (ELF) of the lower respiratory tract normally contains high GSH levels and lung ELF GSH deficiency states can exist, we evaluated the feasibility of augmenting lung ELF GSH levels by (i) administering GSH to sheep i.v. and by direct aerosolization and then (ii) measuring the GSH levels in lung ELF, lung lymph, venous plasma, and urine. When GSH (600 mg) was administered i.v. (n = 11), GSH levels in venous plasma, lung lymph, and ELF rose, but only transiently, suggesting the i.v. route would not deliver adequate GSH to the alveolar epithelial surface. For directly administering GSH to the lung by the aerosol route, in vitro studies were first conducted to show that greater than 50% of a GSH solution could be converted to droplets less than 3 microns in aerodynamic diameter without oxidizing the GSH. To target functional GSH to the lower respiratory tract, an aerosolized solution of GSH (600 mg) was administered to sheep (n = 12). Significantly, the GSH level in ELF increased 7-fold at 30 min (preaerosol, 45.7 +/- 10 microM; 30-min postaerosol, 337 +/- 64 microM; P less than 0.001). The ELF GSH levels remained above baseline at 1 hr (P less than 0.01), returning toward baseline over a 2-hr period. In contrast, GSH levels in lung lymph, venous plasma, and urine were not significantly increased during the period--i.e., aerosol therapy selectively augmented the GSH levels only at the lung epithelial surface. Thus, functional GSH can be delivered by aerosol to directly augment the ELF GSH levels of the lower respiratory tract. Such an approach may prove useful in treating a variety of lung disorders.
Genetic mapping and characterization of sorghum and related crops by means of maize DNA probes.
Authors: Hulbert S H , Richter T E , Axtell J D , Bennetzen J L
Published: May 2006

Cloned DNA fragments from 14 characterized maize genes and 91 random fragments used for genetic mapping in maize were tested for their ability to hybridize and detect restriction fragment length polymorphisms in sorghum and other related crop species. Most DNA fragments tested hybridized strongly to DNA from sorghum, foxtail millet, Johnsongrass, and sugarcane. Hybridization to pearl millet DNA was generally weaker, and only a few probes hybridized to barley DNA under the conditions used. Patterns of hybridization of low-copy sequences to maize and sorghum DNA indicated that the two genomes are very similar. Most probes detected two loci in maize; these usually detected two loci in sorghum. Probes that detected one locus in maize generally detected a single locus in sorghum. However, maize repetitive DNA sequences present on some of the genomic clones did not hybridize to sorghum DNA. Most of the probes tested detected polymorphisms among a group of seven diverse sorghum lines tested; over one-third of the probes detected polymorphism in a single F2 population from two of these lines. Cosegregation analysis of 55 F2 individuals enabled several linkage groups to be constructed and compared with the linkage relationships of the same loci in maize. The linkage relationships of the polymorphic loci in the two species were usually conserved, but several rearrangements were detected.
Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human.
Authors: MacDonald P N , Bok D , Ong D E
Published: May 2006

Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the endothelial cells of the brain microvasculature and within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.