bluuebunny/crossref_metadata_2025 huggingface dataset:
Macromolecular assemblage in the design of a synthetic AIDS vaccine.
Authors: Defoort J P , Nardelli B , Huang W , Ho D D , Tam J P
Published: May 2006

We describe a peptide vaccine model based on the mimicry of surface coat protein of a pathogen. This model used a macromolecular assemblage approach to amplify peptide antigens in liposomes or micelles. The key components of the model consisted of an oligomeric lysine scaffolding to amplify peptide antigens covalently 4-fold and a lipophilic membrane-anchoring group to further amplify noncovalently the antigens many-fold in liposomal or micellar form. A peptide antigen derived from the third variable domain of glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1), consisting of neutralizing, T-helper, and T-cytotoxic epitopes, was used in a macromolecular assemblage model (HIV-1 linear peptide amino acid sequence 308-331 in a tetravalent multiple antigen peptide system linked to tripalmitoyl-S-glycerylcysteine). The latter complex, in liposome or micelle, was used to immunize mice and guinea pigs without any adjuvant and found to induce gp120-specific antibodies that neutralize virus infectivity in vitro, elicit cytokine production, and prime CD8+ cytotoxic T lymphocytes in vivo. Our results show that the macromolecular assemblage approach bears immunological mimicry of the gp120 of HIV virus and may lead to useful vaccines against HIV infection.
Ras proteins are essential and selective for the action of insulin-like growth factor 1 late in the G1 phase of the cell cycle in BALB/c murine fibroblasts.
Authors: Lu K , Campisi J
Published: May 2006

BALB/c 3T3 cells (A31 cells) require the sequential action of growth factors in order to proliferate from a quiescent growth state. Insulin-like growth factor I (IGF-I) is needed late in the G1 phase of the cell cycle, a time at which expression of the c-Ha-ras protooncogene is near maximal. An anti-ras antibody, introduced by microinjection, specifically blocked the ability of IGF-I to stimulate initiation of DNA synthesis. The antibody was specific for IGF-I; it failed to block serum, platelet-derived growth factor, or epidermal growth factor from inducing c-fos mRNA. By contrast, an anti-G alpha-subunit antibody had no effect on IGF-I-stimulated DNA synthesis but inhibited the induction of c-fos mRNA by platelet-derived growth factor or epidermal growth factor. BPA31 cells are tumorigenic A31-derived cells that progress through G1 in the absence of IGF-I. BPA31 cells produced an autocrine IGF-I that was responsible for the loss of late G1 control; the anti-ras antibody arrested the growth of these cells in late G1. The results suggest that ras proteins are essential for an IGF-I-sensitive, G1 control point.
HD-Zip proteins: members of an Arabidopsis homeodomain protein superfamily.
Authors: Schena M , Davis R W
Published: May 2006

Homeobox genes encode a large family of homeodomain proteins in animal systems. To test whether such genes are also abundant in higher plants, degenerate oligonucleotides complementary to sequences encoding the recognition helix (helix three) of the homeodomain were used to screen genomic and cDNA libraries from the plant Arabidopsis thaliana. Analysis of 8 of the 41 cDNAs isolated revealed that each encodes a presumptive homeodomain; interestingly, most of these clones also contain a leucine zipper motif tightly linked to the homeodomain. It is concluded that Arabidopsis encodes a large family of homeodomain proteins, including members that contain a homeodomain/leucine-zipper (HD-Zip) motif.
Chloroplast heteroplasmicity is stabilized by an amber-suppressor tryptophan tRNA(CUA).
Authors: Yu W , Spreitzer R J
Published: May 2006

Photosynthesis-deficient mutants of the green alga Chlamydomonas reinhardtii were previously shown to arise from nonsense mutations within the chloroplast rbcL gene, which encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Photosynthesis-competent revertants of these nonsense mutants have often been found to be stably heteroplasmic, giving rise to both mutant and revertant cells during mitotic or meiotic divisions under nonselective growth conditions. It was proposed that the stable heteroplasmicity might arise from a balanced polymorphism of suppressor and wild-type alleles of a tRNA gene maintained within the polyploid chloroplast genome. In the present study, we have focused on determining the molecular basis for the heteroplasmicity of one such revertant, named R13-3C, which was recovered from the 18-7G amber (UAG) mutant. Restriction-enzyme analysis and DNA sequencing showed that the amber mutation is still present in the rbcL gene of the revertant strain. In contrast, DNA sequencing of the suspected tRNA(Trp) gene of the revertant revealed a mutation that would change its CCA anticodon to amber-specific CUA. This mutation was found to be heteroplasmic, being present in only 70% of the tRNA(Trp) gene copies. Under nonselective conditions, the suppressor mutation was lost from cells that also lost the revertant phenotype. We conclude that stable heteroplasmicity can arise as a balanced polymorphism of organellar alleles. This observation suggests that additional tRNA suppressors may be identified due to their heteroplasmic nature within polyploid genomes.
Endogenous glucocorticoids regulate an inducible cyclooxygenase enzyme.
Authors: Masferrer J L , Seibert K , Zweifel B , Needleman P
Published: May 2006

The effect of endogenous glucocorticoids on the expression of the cyclooxygenase enzyme was studied by contrasting cyclooxygenase expression and prostanoid synthesis in adrenalectomized and sham-adrenalectomized mice with or without the concurrent administration of endotoxin. Peritoneal macrophages obtained from adrenalectomized mice showed a 2- to 3-fold induction in cyclooxygenase synthesis and activity when compared to sham controls. Intravenous injection of a sublethal dose of endotoxin (5 micrograms/kg) further stimulated cyclooxygenase synthesis, resulting in a 4-fold increase in prostaglandin production. Similar cyclooxygenase induction can be achieved in macrophages obtained from normal mice but only after high doses of endotoxin (2.5 mg/kg) that are 100% lethal to adrenalectomized mice. Restoration of glucocorticoids in adrenalectomized animals with dexamethasone completely inhibited the elevated cyclooxygenase and protected these animals from endotoxin-induced death. In contrast, no signs of cyclooxygenase induction were observed in the kidneys of the adrenalectomized mice, even when treated with endotoxin. Dexamethasone did not affect the constitutive cyclooxygenase activity and prostaglandin production present in normal and adrenalectomized kidneys. These data indicate the existence of a constitutive cyclooxygenase that is normally present in most cells and tissues and is unaffected by steroids and of an inducible cyclooxygenase that is expressed only in the context of inflammation by proinflammatory cells, like macrophages, and that is under glucocorticoid regulation. Under normal physiological conditions glucocorticoids maintain tonic inhibition of inducible cyclooxygenase expression. Depletion of glucocorticoids or the presence of an inflammatory stimulus such as endotoxin causes rapid induction of this enzyme, resulting in an exacerbated inflammatory response that is often lethal.
Selective expansion of T cells expressing T-cell receptor variable regions V beta 2 and V beta 8 in Kawasaki disease.
Authors: Abe J , Kotzin B L , Jujo K , Melish M E , Glode M P , Kohsaka T , Leung D Y
Published: May 2006

Kawasaki disease (KD) is an acute vasculitis complicated by the development of coronary artery abnormalities. The etiology of KD is unknown. Based on the observation that KD is associated with marked activation of T cells and monocyte/macrophages, we hypothesized that KD may be caused by a superantigen [e.g., a bacterial toxin that stimulates T cells expressing particular T-cell receptor beta chain variable (V beta) gene segments]. Peripheral blood T cells from patients in the acute and convalescent phases of KD and from various control groups were analyzed for T-cell receptor V beta gene expression by using a quantitative PCR technique and cytofluorographic analysis with available anti-V beta monoclonal antibodies. Patients with acute KD demonstrated significantly elevated levels of circulating V beta 2+ and V beta 8.1+ T cells compared to the other control groups. none of the other 20 V beta populations analyzed by quantitative PCR were found to be significantly elevated. Using flow cytometry, we confirmed a significant elevation of T cells reactive with anti-V beta 8.1 and the lack of change in several other V beta subsets--i.e. V beta 5.1, -5.2, -6.7, and -12. During the convalescence phase of KD, there was a reduction in the abnormal levels of V beta 2+ and V beta 8.1+ T cells. These observations suggest that KD may be caused by a superantigen and may provide insight into the nature of the etiologic agent.
Mutations of human myristoyl-CoA:protein N-myristoyltransferase cause temperature-sensitive myristic acid auxotrophy in Saccharomyces cerevisiae.
Authors: Duronio R J , Reed S I , Gordon J I
Published: May 2006

We have isolated cDNAs encoding human myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) by complementing the nmtl-181 mutation of Saccharomyces cerevisiae, which causes temperature-sensitive myristic acid auxotrophy. Human NMT is derived from a single-copy gene, contains 416 amino acids, is 44% identical to S. cerevisiae NMT (yeast NMT), and can complement the lethal phenotype of an nmtl null mutation. Human and yeast NMTs have overlapping yet distinct protein substrate specificities as judged by a coexpression system that reconstitutes protein N-myristoylation in Escherichia coli. Both enzymes contain a glycine five residues from the C terminus. Gly----Asp or Lys mutagenesis in these orthologous NMTs produces marked reductions in their activities in E. coli as well as temperature-sensitive myristic acid auxotrophy in S. cerevisiae. These results indicate highly conserved structure-function relationships in vivo and underscore the usefulness of these functional assays for identifying factors that regulate protein N-myristoylation in mammalian systems.
The Antennapedia-type homeobox genes have evolved from three precursors separated early in metazoan evolution.
Authors: Schubert F R , Nieselt-Struwe K , Gruss P
Published: May 2006

The developmental control genes containing an Antennapedia-type homeobox are clustered in insects and vertebrates. The evolution of these genes was studied by the construction of evolutionary trees and by statistical geometry in sequence space. The comparative analysis of the homeobox sequences reveals the subdivision of the Antennapedia-type homeobox genes into three classes early in metazoan evolution. This observation suggests an important function of these genes even in the most primitive metazoans. Subsequent duplication events generated a cluster of at least five homeobox genes in the last common ancestor of insects and vertebrates. These genes later independently gave rise to the 13 groups of paralogous genes in vertebrates and to the 11 Antennapedia-type genes in the Drosophila complexes.
Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues.
Authors: Kobayashi M , Izui H , Nagasawa T , Yamada H
Published: May 2006

Indole-3-acetic acid is the major auxin in most plants. In Cruciferae, including Brassicaceae, indole-3-acetic acid is synthesized from indole-3-acetonitrile by nitrilase, after indole-3-acetonitrile is formed from tryptophan via indole-3-acetaldoxime or indole glycosinolates as the intermediate. We cloned and sequenced the gene for nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of indole-3-acetonitrile to indole-3-acetic acid, from Alcaligenes faecalis JM3. The amino acid sequence deduced from the nucleotide sequence of the nitrilase gene shows 34.7% identity with that of Klebsiella ozaenae nitrilase. A DNA clone containing the nitrilase gene expressed the active enzyme in Escherichia coli with excellent yield. Among five cysteine residues (Cys-40, Cys-115, Cys-162, Cys-163, and Cys-218) in the Alcaligenes nitrilase, only Cys-163 was conserved at the corresponding position in the Klebsiella nitrilase. Two mutant enzymes, in which Cys-162 and Cys-163 were replaced with Asn and Ala, respectively, were constructed by site-directed mutagenesis. A 35% increase of the specific activity and a large reduction of the Km for thiophene-2-acetonitrile (which was used as a standard substrate for the nitrilase) were observed in the Cys-162-->Asn mutant enzyme. The Cys-163-->Ala mutation resulted in complete loss of nitrilase activity, clearly indicating that Cys-163 is crucial for the activity and Cys-162 could not provide the catalytic function of Cys-163.
Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids.
Authors: Allenby G , Bocquel M T , Saunders M , Kazmer S , Speck J , Rosenberger M , Lovey A , Kastner P , Grippo J F , Chambon P
Published: May 2006

The binding of endogenous retinoids and stereoisomers of retinoic acid (RA) to the retinoid nuclear receptors, RA receptor (RARs) and retinoid X receptors (RXRs), was characterized using nucleosol preparations from transiently transfected COS-1 cells. Among several stereoisomers of RA tested, including 7-cis-, 9-cis-, 11-cis-, 13-cis-, and all-trans-RA, only 9-cis-RA effectively competes with 9-cis-[3H]RA binding to the RXRs. Additionally, the endogenous retinoid trans-didehydro-RA (t-ddRA) does not interact with RXRs, whereas the 9-cis form of ddRA competes effectively. RXRs (alpha, beta, and gamma) bind 9-cis-RA with dissociation constants (Kd) of 15.7, 18.3, and 14.1 nM, respectively. In contrast to the selectivity of RXRs for 9-cis-RA, RARs bind both t-RA and 9-cis-RA with high affinity, exhibiting Kd values in the 0.2-0.7 nM range for both ligands. Unlike RARs, the cellular RA binding proteins CRABPI or CRABPII bind t-RA but do not bind 9-cis-RA. Consistent with the binding data, 9-cis-RA and 9-cis-ddRA transcriptionally activate both GAL4-RXR and GAL4-RAR chimeric receptors with EC50 values of 3-20 nM for 9-cis-RA and 9-cis-ddRA, whereas t-RA and t-ddRA efficiently activate only GAL4-RAR chimeric receptors. Thus, 9-cis forms of endogenous retinoids can contribute to the pleiotropic effects of retinoids by interacting with both the RARs and RXRs.
Activation of glucose transport by a natural mutation in the human insulin receptor.
Authors: Longo N , Langley S D , Griffin L D , Elsas L J
Published: May 2006

Naturally occurring mutations in the insulin receptor gene cause heritable severe insulin resistance. These mutations usually impair insulin receptor signaling in cells cultured from affected individuals. However, fibroblasts cultured from a patient with intrauterine growth restriction and severe insulin resistance (leprechaun Atl-1) had normal amounts of insulin receptor protein and defective insulin binding but constitutive activation of insulin-receptor autophosphorylation and kinase activity and of glucose transport. In the same fibroblasts, growth was impaired. Homozygosity for a mutation in the insulin receptor gene was suspected, since he inherited identical DNA haplotypes for this gene from both related parents. Here we report that the proband was homozygous and both parents were heterozygous for a point mutation in the insulin receptor gene converting the Arg86 codon (CGA) to Pro (CCA) (R86P). The R86P substitution is contiguous to the hydrophobic beta-sheet of the receptor alpha subunit implicated by DeMeyts et al. [DeMeyts, P., Gu, J.-L., Shymko, R. M., Kaplan, B. E., Bell, G. I. & Whittaker, J. (1990) Mol. Endocrinol. 4, 409-416] in the binding of aromatic residues of the insulin molecule. The R86P mutant insulin receptor cDNA was inserted into a plasmid under control of a simian virus 40 promoter and transfected into Chinese hamster ovary (CHO) cells. In contrast with fibroblasts from patient Atl-1, which had normal insulin receptor processing, CHO cells stably transfected with the R86P mutant cDNA (CHO-R86P) had altered posttranslational processing. The R86P mutant receptor failed to bind insulin but caused a significant increase in basal glucose transport in CHO cells. As in fibroblasts cultured from the patient, the R86P mutant insulin receptor did not stimulate growth in transfected CHO cells. These results suggest that the R86P mutation in the insulin receptor activates glucose transport without promoting cell growth and that distinct cell types process this mutant insulin receptor differently.
Steroids and their conjugates in the mammalian brain.
Authors: Mathur C , Prasad V V , Raju V S , Welch M , Lieberman S
Published: May 2006

Five steroids--3 beta-hydroxypregn-5-en-20-one (pregnenolone; P), 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-AP), 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-AP), 3 beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone; D), and 3 beta-hydroxy-5 alpha-androstan-17-one (EpiA)--were extracted from the brains of adult male rats, rabbits, and dogs. The steroids exist in this organ as unconjugated compounds and as sulfates, lipoidal esters, and sulfolipids. The techniques for separating these four classes of steroids from each other and for separating the five steroids from each other are described. In all cases, the steroids were identified by their retention time (Rt) on HPLC, their Rt by gas chromatography, and by selected ion monitoring of their mass spectra. The latter were also used for quantification. In their reaction toward organic bases, the sulfolipid conjugates resemble previously described sulfolipids of cholesterol and sitosterol. These conjugates are relatively abundant in brain, particularly those of P and D, and this suggests that, in the search for the physiological significance of these brain constituents, these conjugates warrant attention.
Extrathymic differentiation of T lymphocytes and natural killer cells from human embryonic liver precursors.
Authors: Poggi A , Sargiacomo M , Biassoni R , Pella N , Sivori S , Revello V , Costa P , Valtieri M , Russo G , Mingari M C
Published: May 2006

Liver cells were isolated on Ficoll/Hypaque gradients from embryos or fetuses at 6-10 weeks of gestation; 2-20% of the cells expressed CD45 or HLA class I surface antigens and 2-6% expressed CD7. Other T- or natural-killer (NK)-cell-lineage-specific markers were undetectable. Liver-cell suspensions cultured in the presence of phytohemagglutinin and recombinant interleukin 2 gave rise to large proportions of CD3+ lymphocytes expressing either alpha/beta or gamma/delta T-cell receptors. This occurred not only in bulk cultures but also when cells were cloned under limiting dilution conditions. Importantly, these figures were obtained also in embryos at 6-8 weeks of gestation, which is before colonization of the thymic rudiment by T-cell precursors. When the same liver-cell suspensions were cultured in the presence of irradiated H9 cells and recombinant interleukin 2 (either in bulk cultures or under cloning conditions), large proportions of cells (or clones) expressed surface CD16 and CD56 antigens and displayed a strong cytolytic activity against both NK-sensitive (K562) and NK-resistant (M14) target cells. In addition, liver-derived T or NK cells expressed functional receptor molecules since they could be activated via either CD3/T-cell receptor or CD16 surface antigens, respectively. Further fractionation of liver cells on the basis of CD45 antigen expression indicated that only CD45+ cells could give rise to T or NK cells in culture. Thus, CD45 can be used as a marker for identification of an early liver-cell population containing T- and NK-cell precursors. That T or NK cells were derived from male embryos and not from the mother was shown by PCR amplification of X and Y chromosomal sequences. Our present data may offer an in vitro model for extrathymic embryonic T-cell maturation that can be used to examine fundamental aspects of human T-cell development and function.
Methylation induced premeiotically in Ascobolus: coextension with DNA repeat lengths and effect on transcript elongation.
Authors: Barry C , Faugeron G , Rossignol J L
Published: May 2006

The effect of duplications of gene fragments on the triggering of DNA methylation induced premeiotically (MIP) was studied in the sexual progeny of strains harboring, in addition to the resident met2 gene, a fragment of this gene inserted at an ectopic position. Cytosine methylation of the resident gene was checked for each of the eight duplications tested. Methylation was always found and it was coextensive with the length of the duplications. Silencing of the resident gene was triggered by duplications of segments corresponding to the region 5' to the open reading frame, to only the open reading frame, or to segments beginning 0.87-1.2 kb downstream from the transcription start. Silencing was accompanied by either the absence of transcripts or the presence of truncated transcripts, which suggests that methylation acts on transcript elongation.
Intracellular calcium increases with hyperactivation in intact, moving hamster sperm and oscillates with the flagellar beat cycle.
Authors: Suarez S S , Varosi S M , Dai X
Published: May 2006

At some time before fertilization, mammalian sperm undergo a change in movement pattern, termed hyperactivation. There is evidence that hyperactivation offers an advantage to sperm for detaching from the oviductal mucosa, for penetrating viscoelastic substances in the oviduct, and for penetrating the zona pellucida. Hyperactivation is known to require extracellular calcium, but little else is known about the mechanisms by which calcium affects sperm movement. The calcium-sensitive fluorescent dye indo-1 was used to follow intracellular calcium levels ([Ca2+]i) in individual moving sperm. Sperm were loaded with 10 microM of the acetoxymethyl ester form of the dye and then rinsed. The dye was excited at 340 nm by using a filtered xenon stroboscope, and images at the 405-nm and 490-nm excitation maxima were simultaneously digitized at 30 per sec for 2.1 sec. [Ca2+]i was significantly higher in the acrosomal and postacrosomal regions of the head and in the flagellar midpiece (the principal piece could not be measured) in hyperactivated than in nonhyperactivated sperm (P < 0.0001). [Ca2+]i oscillations were detected in the proximal half of the midpiece that were identical in frequency to the flagellar-beat-cycle frequency in 12 of 17 hyperactivated sperm (median, 3.5 Hz). Rapid [Ca2+]i oscillations were also detected in the acrosomal and postacrosomal regions, as well as in the distal midpiece. Oscillations were not eliminated by dampening the flagellar bending with methyl cellulose. The [Ca2+]i oscillations detected in sperm are significantly more rapid than oscillations detected in other cell types.
Amyloid beta-protein as a substrate interacts with extracellular matrix to promote neurite outgrowth.
Authors: Koo E H , Park L , Selkoe D J
Published: May 2006

Progressive deposition of amyloid beta-protein (A beta) in brain parenchyma and blood vessels is a characteristic feature of Alzheimer disease. Recent evidence suggests that addition of solubilized synthetic A beta to medium may produce toxic or trophic effects on cultured hippocampal neurons. Because soluble A beta may not accumulate in significant quantities in brain, we asked whether immobilized A beta peptide as a substrate alters neurite outgrowth from cultured rat peripheral sensory neurons. This paradigm may closely mimic the conditions in Alzheimer disease brain tissue, in which neurites contact insoluble, extracellular aggregates of beta-amyloid. We detected no detrimental effects of A beta substrate on neurite outgrowth. Rather, A beta in combination with low doses of laminin or fibronectin enhanced neurite out-growth from these neuronal explants. Our results suggest that insoluble A beta in the cerebral neuropil may serve as a neurite-promoting matrix, perhaps explaining the apparent regenerative response of neurites observed around amyloid plaques in Alzheimer disease. Moreover, in concert with the recent discovery of A beta production by cultured neurons, our data suggest that A beta plays a normal physiological role in brain by complexing with the extracellular matrix.
Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an antarctic bacterium.
Authors: Langin D , Laurell H , Holst L S , Belfrage P , Holm C
Published: May 2006

The human hormone-sensitive lipase (HSL) gene encodes a 786-aa polypeptide (85.5 kDa). It is composed of nine exons spanning approximately 11 kb, with exons 2-5 clustered in a 1.1-kb region. The putative catalytic site (Ser423) and a possible lipid-binding region in the C-terminal part are encoded by exons 6 and 9, respectively. Exon 8 encodes the phosphorylation site (Ser551) that controls cAMP-mediated activity and a second site (Ser553) that is phosphorylated by 5'-AMP-activated protein kinase. Human HSL showed 83% identity with the rat enzyme and contained a 12-aa deletion immediately upstream of the phosphorylation sites with an unknown effect on the activity control. Besides the catalytic site motif (Gly-Xaa-Ser-Xaa-Gly) found in most lipases, HSL shows no homology with other known lipases or proteins, except for a recently reported unexpected homology between the region surrounding its catalytic site and that of the lipase 2 of Moraxella TA144, an antarctic psychrotrophic bacterium. The gene of lipase 2, which catalyses lipolysis below 4 degrees C, was absent in the genomic DNA of five other Moraxella strains living at 37 degrees C. The lipase 2-like sequence in HSL may reflect an evolutionarily conserved cold adaptability that might be of critical survival value when low-temperature-mobilized endogenous lipids are the primary energy source (e.g., in poikilotherms or hibernators). The finding that HSL at 10 degrees C retained 3- to 5-fold more of its 37 degrees C catalytic activity than lipoprotein lipase or carboxyl ester lipase is consistent with this hypothesis.
Kinetics of p56lck and p60src Src homology 2 domain binding to tyrosine-phosphorylated peptides determined by a competition assay or surface plasmon resonance.
Authors: Payne G , Shoelson S E , Gish G D , Pawson T , Walsh C T
Published: May 2006

Src homology 2 (SH2) domains are phosphotyrosine-binding modules found within various signal-transducing proteins. We have determined by 125I competition assay and surface plasmon resonance that the SH2 domains of Src and Lck bind to a variety of phosphopeptides with similar affinity and specificity. Both bound with highest affinity [Kd(app) approximately 3.7 nM; ka = 2.4 x 10(5) M-1 x s-1; kd = 1.2 x 10(-3) s-1] a phosphopeptide having a Tyr(P)-Glu-Glu-Ile motif found in the hamster polyomavirus middle-sized tumor antigen. Intermediate affinity (5- to 40-fold lower) was observed with phosphopeptides corresponding to the regulatory domains of Src and Lck, containing Tyr527 and Tyr505, respectively. Lowest affinity (80- to 300-fold lower) was observed with phosphopeptides corresponding to phosphorylated tyrosines of GTPase-activating protein, insulin receptor substrate 1, and SH2 domain-containing protein-tyrosine-phosphatase 1.
Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages.
Authors: Kovacsovics-Bankowski M , Clark K , Benacerraf B , Rock K L
Published: May 2006

Antigens in extracellular fluids can be processed and presented with major histocompatibility complex (MHC) class I molecules by a subset of antigen presenting cells (APCs). Chicken egg ovalbumin (Ova) linked to beads was presented with MHC class I molecules by these cells up to 10(4)-fold more efficiently than soluble Ova. This enhanced presentation was observed with covalently or noncovalently linked Ova and with beads of different compositions. A key parameter in the activity of these conjugates was the size of the beads. The APC that is responsible for this form of presentation is a macrophage. These cells internalize the antigen constructs through phagocytosis, since cytochalasin B inhibited presentation. Processing of the antigen and association with MHC class I molecules appears to occur intracellularly as presentation was observed under conditions where there was no detectable release of peptides into the extracellular fluids. When injected in vivo in C57BL/6 mice, Ova-beads, but not soluble Ova, primed CD4- CD8+ cytotoxic T lymphocytes (CTLs). Similar results were obtained in BALB/c mice immunized with beta-galactosidase-beads. The implications of these findings for development of nonliving vaccines that stimulate CTL immunity are discussed.
The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: a potential role for cellular raf kinases in neuronal growth factor signal transduction.
Authors: Wood K W , Qi H , D'Arcangelo G , Armstrong R C , Roberts T M , Halegoua S
Published: May 2006

The neuron-like differentiation of PC12 cells is induced by nerve growth factor (NGF) through stimulation of a membrane-bound protooncoprotein signaling pathway containing the NGF receptor Trk, the tyrosine kinase Src, and the GTP-binding protein Ras. The Raf-1 and B-raf protooncogenes encode cytoplasmic serine/threonine kinases that are stimulated by NGF in a Ras-dependent manner. To investigate the possible roles of cytoplasmic Raf kinases in eliciting neuronal differentiation, we have expressed the activated Raf-1 oncogene in PC12 cells. Expression of the raf oncogene results in the elaboration of a neuron-like phenotype, including neurite growth and the induction of the NGF-responsive genes NGFI-A and transin. The actions of activated Raf-1 and NGF are not additive. Furthermore, activated Raf-1 oncoprotein can prime cells for transcription-independent neurite growth by NGF and can elicit rapid neurite growth from NGF-primed cells. Our data indicate that the pathways utilized by NGF and activated raf to effect PC12 differentiation overlap and lead to the suggestion that cellular raf kinase activities play significant roles in transducing the differentiating signals of neuronal growth factors.
Three-dimensional structures of avidin and the avidin-biotin complex.
Authors: Livnah O , Bayer E A , Wilchek M , Sussman J L
Published: May 2006

The crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and of its complex with biotin have been determined to 2.6 and 3.0 A, respectively. The structures reveal the amino acid residues critical for stabilization of the tetrameric assembly and for the exceptionally tight binding of biotin. Each monomer is an eight-stranded antiparallel beta-barrel, remarkably similar to that of the genetically distinct bacterial analog streptavidin. As in streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. There are, however, some differences. The presence of additional hydrophobic and hydrophilic groups in the binding site of avidin (which are missing in streptavidin) may account for its higher affinity constant. Two amino acid substitutions are proposed to be responsible for its susceptibility to denaturation relative to streptavidin. Unexpectedly, a residual N-acetylglucosamine moiety was detected in the deglycosylated avidin monomer by difference Fourier synthesis.
Transgenic mice expressing the tumor marker germ cell alkaline phosphatase: an in vivo tumor model for human cancer antigens.
Authors: Narisawa S , Smans K A , Avis J , Hoylaerts M F , Millán J L
Published: May 2006

We have generated a series of transgenic mouse lines harboring the entire human germ cell alkaline phosphatase (GCAP) gene linked to progressively longer sequences of flanking DNA. A 450-bp promoter sequence directs the expression of GCAP to the intestine and endothelial cells, while a 5' sequence of 1.7 kb directs GCAP expression to the spermatogenic lineage and to the eight-cell through the blastocyst stage of preimplantation development. The expression of GCAP in these FVB/N transgenic mice induces a cellular immune tolerance to GCAP. When mouse fibrosarcoma MO4 cells (C3H derived), stably transfected with the cloned GCAP gene, were injected s.c. in nontransgenic control (C3H x FVB/N) hybrid mice, GCAP-positive tumor cells were rejected. However, when GCAP-expressing transgenic (C3H x FVB/N) hybrid mice were challenged with these cells, GCAP-positive tumors developed. Tumors also developed in the transgenic hybrid mice upon injection of MO4 cells transfected with the highly homologous placental alkaline phosphatase (PLAP) cDNA in spite of the presence in PLAP of 10 amino acids that are different from the corresponding residues in GCAP. These GCAP transgenic mice will allow the study of the immune response associated with the repeated administration of conjugated or derivatized anti-GCAP and anti-PLAP monoclonal antibodies. They will also enable evaluation of the therapeutic potential of bifunctional antibodies for T-cell recruitment and destruction of GCAP/PLAP-producing tumor cells.
Expression cloning of an ATP receptor from mouse neuroblastoma cells.
Authors: Lustig K D , Shiau A K , Brake A J , Julius D
Published: May 2006

Extracellular ATP activates cell-surface metabotropic and ionotropic nucleotide (P2) receptors in vascular, neural, connective, and immune tissues. These P2 receptors mediate a wealth of physiological processes, including nitric oxide-dependent vasodilation of vascular smooth muscle and fast excitatory neurotransmission in sensory afferents. Although ATP is now recognized as a signaling molecule, the cellular and molecular mechanisms underlying its actions have been difficult to study due to the absence of selective P2 receptor antagonists and cloned receptor genes. Nonetheless, five mammalian P2 receptor subtypes have been tentatively assigned based solely on agonist specificity and signaling properties. Here we report the cloning of a mouse cDNA encoding a P2 receptor that shares striking homology with several G protein-coupled peptide receptors. When expressed in Xenopus laevis oocytes, the cloned receptor resembles a metabotropic P2U receptor; activation by either ATP or UTP elicits the mobilization of intracellular calcium. mRNA encoding the P2U purinergic receptor is found in neural and nonneural tissues.
Identification of two missense mutations in a dihydrolipoamide dehydrogenase-deficient patient.
Authors: Liu T C , Kim H , Arizmendi C , Kitano A , Patel M S
Published: May 2006

The molecular basis of dihydrolipoamide dehydrogenase (E3; dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) deficiency in an E3-deficient patient was studied. Fibroblasts cultured from the patient contained only approximately 6% of the E3 activity of cells from a normal subject. Western and Northern blot analyses indicated that, compared to control cells, the patient's cells had a reduced amount of protein but normal amounts of E3 mRNA. Direct sequencing of E3 cDNA derived from the patient's RNA as well as each of the subclones of the cDNA revealed that the patient had two substitution mutations in the E3 coding region. One mutation changed a single nucleotide from A to G, resulting in substitution of Glu (GAA) for Lys-37 (AAA). The other point mutation was a nucleotide change from C to T, resulting in the substitution of Leu (CTG) for Pro-453 (CCG). These mutations appear to be significant in that they alter the active site and possibly the binding of FAD.
Association of m1 and m2 muscarinic receptor proteins with asymmetric synapses in the primate cerebral cortex: morphological evidence for cholinergic modulation of excitatory neurotransmission.
Authors: Mrzljak L , Levey A I , Goldman-Rakic P S
Published: May 2006

Muscarinic m1 receptors traditionally are considered to be postsynaptic to cholinergic fibers, while m2 receptors are largely presynaptic receptors associated with axons. We have examined the distribution of these receptor proteins in the monkey cerebral cortex and obtained results that are at odds with this expectation. Using immunohistochemistry with specific antibodies to recombinant m1 and m2 muscarinic receptor proteins, we have demonstrated that both m1 and m2 receptors are prominently associated with noncholinergic asymmetric synapses as well as with the symmetric synapses that characterize the cholinergic pathways in the neocortex. At asymmetric synapses, both m1 and m2 receptor immunoreactivity is observed postsynaptically within spines and dendrites; the m2 receptor is also found in presynaptic axon terminals which, in the visual cortex, resemble the parvicellular geniculocortical pathway. In addition, m2 labeling was also found in a subset of nonpyramidal neurons. These findings establish that the m2 receptor is located postsynaptically as well as presynaptically. The association of m1 and m2 receptors with asymmetric synapses in central pathways, which use excitatory amino acids as neurotransmitters, provides a morphological basis for cholinergic modulation of excitatory neurotransmission.
Alpha 2-adrenergic inhibition of Cl- transport by opercular epithelium is mediated by intracellular Ca2+.
Authors: Marshall W S , Bryson S E , Garg D
Published: May 2006

We isolated the opercular epithelium of sea-water killifish (Fundulus heteroclitus) to study the mediation of catecholamine inhibition of Cl- secretion. The receptors are alpha 2-adrenergic, as they have a high affinity for the alpha 2-adrenergic agonist clonidine over phenylephrine and clonidine action is blocked by yohimbine. Pertussis toxin and indomethacin did not block the clonidine effect; hence inhibitory guanine nucleotide-binding proteins (Gi proteins) and prostaglandins (respectively) are not involved. Intracellular pH (pHi) of single chloride cells was measured microspectrofluorometrically and resting pHi was 7.22 +/- 0.03. However, pHi was unaffected by clonidine; hence pHi and Na+/H+ exchange are not involved. The lipoxygenase inhibitors nordihydroguaiaretic acid and baicalein and the lipoxygenase products (12S)- and (12R)-12-hydroxyeicosatetraenoic acid stimulated Cl- secretion. Protein kinase C is an unlikely site of action because the diacylglycerol kinase inhibitor R59022 had no effect alone and did not block the clonidine effect. Ionomycin (1 microM) in normal but not low-Ca2+ solutions mimicked the action of clonidine and both inhibitions were reversible by isoproterenol. Thapsigargin, a releaser of intracellular Ca2+, inhibited Cl- secretion and this effect was reduced in low-Ca2+ solutions. Low-Ca2+ solutions also blunted but did not block entirely the clonidine response, indicating that the primary Ca2+ release was from intracellular stores. Whereas alpha 1-adrenergic receptors commonly act via the Ca2+/inositol trisphosphate pathway, to our knowledge this is the first report of a Ca(2+)-mediated alpha 2-adrenergic response in a nonmammalian vertebrate.
Residues crucial for Ras interaction with GDP-GTP exchangers.
Authors: Segal M , Willumsen B M , Levitzki A
Published: May 2006

Cdc25 is essential for Ras-mediated activation of adenylyl cyclase in the yeast Saccharomyces cerevisiae. This protein acts by catalyzing GDP-GTP exchange on yeast Ras. Harvey (Ha) ras expressed in S. cerevisiae is also recognized by both Cdc25 and Sdc25, a yeast homolog of Cdc25. Thus it is feasible to examine molecular aspects of mammalian Ras modulation by Cdc25 using the RAS/cAMP pathway in yeast as a model system. Here, we describe mutational analysis of Ha-ras for the identification of residues critical for the ability of Ras to interact with Cdc25 and related guanine nucleotide-release proteins. Mutations within codons 97-108 impaired Ras-mediated activation of adenylyl cyclase in the presence but not in the absence of mammalian GTPase-activating protein. Such mutations, therefore, affected the ability of Ras to undergo GDP-GTP exchange catalyzed by the guanine nucleotide exchanger without preventing Ras activation of the effector. Similar mutations were previously shown to impair the ability of c-ras to transform mammalian cells while having a less drastic effect on v-ras.
Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65.
Authors: Chang A , Rose M D , Slayman C W
Published: May 2006

We have developed two independent assays to study the integration, folding, and intracellular transport of the polytopic plasma membrane H(+)-ATPase in yeast. To follow folding, controlled trypsinolysis was used to distinguish between the E1 conformation of the ATPase (favored in the presence of ADP) and the E2 conformation (favored in the presence of vanadate). By this criterion, wild-type ATPase appears to recognize its ligands and assume distinct conformations within a short time after its biosynthesis. To follow intracellular transport, we have exploited the fact that export of newly synthesized ATPase from the endoplasmic reticulum is accompanied by kinase-mediated phosphorylation, leading to a shift in electrophoretic mobility. Because proper folding is required for transport from the endoplasmic reticulum, the mobility shift also serves as a convenient bioassay for correct folding. As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle. Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed. By contrast, in a sec65 mutant, the folding and intracellular transport of newly synthesized ATPase were delayed. Our data suggest that conformational maturation of the ATPase is a rapid process in wild-type cells and that membrane integration mediated by signal recognition peptide is important for the proper folding of this polytopic protein.
Polar/apolar compounds induce leukemia cell differentiation by modulating cell-surface potential.
Authors: Arcangeli A , Carlà M , Del Bene M R , Becchetti A , Wanke E , Olivotto M
Published: May 2006

The mechanism of action of polar/apolar inducers of cell differentiation, such as dimethyl sulfoxide and hexamethylene-bisacetamide, is still obscure. In this paper evidence is provided that their effects on murine erythroleukemia cells are modulated by various extracellular cations as a precise function of the cation effects on membrane surface potential. The interfacial effects of the inducers were directly measured on the charged electrode, showing that both dimethyl sulfoxide and hexamethylene-bisacetamide, at the effective concentrations for cell differentiation and within the physiological range of charge density, adsorb at the charged surface and produce a potential shift. A linear correlation was found between this shift and the inducer effects on cell differentiation. Besides offering a different interpretation of the mechanism of action of the inducers, these findings indicate that surface potential has a signaling function. They may also be relevant to cancer treatments based on tumor-cell commitment to terminal differentiation.
Crypt/villus site of substrate-dependent regulation of mouse intestinal glucose transporters.
Authors: Ferraris R P , Diamond J M
Published: May 2006

The intestinal epithelium is in a constant state of turnover, with cells differentiating at the crypts and then migrating toward the tips of the villi. Does substrate-dependent regulation of intestinal Na+/D-glucose cotransporters occur only in crypt cells, or can transport activity be subsequently reprogrammed in mature enterocytes? We used in situ, glucose-protectable specific phlorizin binding to determine site density of brush border glucose transporters in enterocytes fractionated along the crypt/villus axis of mice that were killed shortly after drastic changes in carbohydrate levels of their diets. Dietary carbohydrate-induced changes in site density of specific phlorizin binding initially appeared only in crypt cells before spreading, over the course of several days, to the villus tips. Thus, only crypt cells perceive the signal for glucose transporter regulation, and the observed time lag of diet-induced changes in intestinal glucose uptake is due mainly to cell migration times.
chiro-inositol deficiency and insulin resistance: a comparison of the chiro-inositol- and the myo-inositol-containing insulin mediators isolated from urine, hemodialysate, and muscle of control and type II diabetic subjects.
Authors: Asplin I , Galasko G , Larner J
Published: May 2006

chiro- and myo-Inositols are major components of the two inositol phosphoglycan mediators of insulin action. Previous work in this laboratory has shown hypo-chiro-inositoluria in type II diabetic subjects and decreased chiro-inositol in mediator prepared from skeletal-muscle biopsies of Pima Indian diabetic subjects together with increased myo-inositol concentrations. Because mediator bioactivity was not previously examined, we decided to isolate the two types of insulin mediator from hemodialysate, urine, and autopsy muscle to investigate their bioactivity in control and type II diabetic subjects. Human mediator fractions were isolated at pH 2.0 and pH 1.3 from hemodialysate, urine, and autopsy muscle of type II diabetic subjects and nondiabetic control subjects. Mediators were assayed for bioactivity, and the relative chiro-inositol/myo-inositol concentration ratio was determined for the mediator pH 2.0 samples by using HPLC or GC/MS. Regardless of source, the chiro-inositol-containing mediator pH 2.0 fractions from type II diabetic subjects were markedly less active than those from controls (50% or less) (P < 0.05). In addition, the chiro-inositol/myo-inositol ratio in samples from type II subjects was significantly reduced (1/3-1/9) compared with controls (P < 0.05 for hemodialysate and P < 0.01 for muscle samples). In contrast, no difference in bioactivity was seen in myo-inositol-containing mediator pH 1.3 samples isolated from the same type II diabetic and control subjects. In type II diabetes there is a generalized deficiency of chiro-inositol mediator in the body in terms of both decreased chiro-inositol mediator (pH 2.0) bioactivity and chiro-inositol content.
Thalidomide inhibits the replication of human immunodeficiency virus type 1.
Authors: Makonkawkeyoon S , Limson-Pobre R N , Moreira A L , Schauf V , Kaplan G
Published: May 2006

Thalidomide, a selective inhibitor of tumor necrosis factor alpha (TNF-alpha) synthesis, suppresses the activation of latent human immunodeficiency virus type 1 (HIV-1) in a monocytoid (U1) line. The inhibition is dose dependent and occurs after exposure of the cells to recombinant TNF-alpha, phorbol myristate acetate, lipopolysaccharide, and other cytokine combinations. Associated with HIV-1 inhibition is a reduction in agonist-induced TNF-alpha protein and mRNA production. Thalidomide inhibition of virus replication in the phorbol myristate acetate- and recombinant TNF-alpha-stimulated T-cell line ACH-2 is not observed. The presence of thalidomide also inhibits the activation of virus in the peripheral blood mononuclear cells of 16 out of 17 patients with advanced HIV-1 infection and AIDS. These results suggest the use of thalidomide in a clinical setting to inhibit both virus replication and the TNF-alpha-induced systemic toxicity of HIV-1 and opportunistic infections.