bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of cAMP levels and of protein kinase activity by tumor necrosis factor and interleukin 1 in human fibroblasts: role in the induction of interleukin 6.
Authors: Zhang Y H , Lin J X , Yip Y K , Vilcek J
Published: May 2006

Although tumor necrosis factor (TNF) and interleukin 1 (IL-1) affect many cell functions, the molecular mechanisms of TNF and IL-1 action are not understood. Our present study shows that exposure of human FS-4 fibroblasts to TNF or IL-1 caused a rapid accumulation of intracellular cAMP and an increase in protein kinase activity. Intracellular cAMP levels peaked 3-5 min after the addition of TNF or IL-1 and returned to basal level by 15 min. Increased phosphorylation of histone HII-B protein was demonstrated with extracts prepared from TNF- or IL-1-treated cells, suggesting an increase in cAMP-dependent protein kinase activity. No evidence was obtained for protein kinase C activation in TNF-treated FS-4 cells. TNF, IL-1, and forskolin all stimulated interleukin 6 (IL-6) mRNA levels in FS-4 cells. The protein kinase inhibitor H-8, inhibiting preferentially cAMP-dependent kinase activity, reduced forskolin-stimulated IL-6 mRNA induction more strongly than TNF- or IL-1-driven IL-6 mRNA induction. These results suggest that activation of cAMP-dependent protein kinase by TNF and IL-1 is important in some actions of these cytokines. In addition, our data on IL-6 induction by TNF and IL-1 suggest that other, yet unidentified, signal transduction mechanisms contribute to TNF and IL-1 actions on gene expression in human fibroblasts.
Human immunodeficiency virus type 1 has an additional coding sequence in the central region of the genome.
Authors: Matsuda Z , Chou M J , Matsuda M , Huang J H , Chen Y M , Redfield R , Mayer K , Essex M , Lee T H
Published: May 2006

Eight coding regions designated gag, pol, env, sor, R, tat, art/trs, and 3' orf have been identified in the genome of the human immunodeficiency virus type 1 (HIV-1). Several other open reading frames have the potential to encode additional viral proteins. In this study, we show that HIV-1 has another coding sequence whose product is expressed during natural infection. Unlike antibody to other HIV-1 proteins, the prevalence of antibody to the product encoded by this region is elevated in patients with acquired immune deficiency syndrome (AIDS). Because no analogous coding region has been identified in HIV-2, the antibody to the product of this coding region may serve as a marker to distinguish infection with HIV-1 from infection with HIV-2.
Human adrenodoxin reductase: two mRNAs encoded by a single gene on chromosome 17cen----q25 are expressed in steroidogenic tissues.
Authors: Solish S B , Picado-Leonard J , Morel Y , Kuhn R W , Mohandas T K , Hanukoglu I , Miller W L
Published: May 2006

Adrenodoxin reductase is a mitochondrial flavoprotein that receives electrons from NADPH, thus initiating the electron-transport chain serving mitochondrial cytochromes P450. We have cloned and sequenced two human adrenodoxin reductase cDNAs that differ by the presence of six additional codons in the middle of one clone. The sequence in this region indicates that these six extra codons arise by alternative splicing of the pre-mRNA. Southern blot hybridization patterns of human genomic DNA cut with four restriction enzymes indicate that the human genome has only one gene for adrenodoxin reductase. Analysis of a panel of mouse-human somatic cell hybrids localized this gene to chromosome 17cen----q25. The alternatively spliced mRNA containing the six extra codons represents 10-20% of all adrenodoxin reductase mRNA. The expression of the adrenodoxin reductase gene may be stimulated by pituitary tropic hormones acting through cAMP, but its response is quantitatively much less than the responses of P450scc and adrenodoxin.
Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22.
Authors: Tsai-Pflugfelder M , Liu L F , Liu A A , Tewey K M , Whang-Peng J , Knutsen T , Huebner K , Croce C M , Wang J C
Published: May 2006

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.
Cholera toxin potentiates IgE-coupled inositol phospholipid hydrolysis and mediator secretion by RBL-2H3 cells.
Authors: McCloskey M A
Published: May 2006

Binding of polyvalent antigens to IgE present in Fc epsilon receptors on the surface of mast cells and the RBL-2H3 cell line triggers the exocytotic release of allergic mediators. Preincubation of RBL-2H3 cells with cholera toxin (CT) was found to potentiate greater than or equal to 2- to 3-fold the rate and final amount of antigen-induced secretion of [3H]serotonin and N-acetyl beta-D-glucosaminidase. This was accompanied by a more variable increase in the initial rate of antigen-triggered formation of inositol phosphates. The holotoxin was required for potentiation, as neither the A nor the B subunit was effective when added separately. Four observations indicate that cAMP was not the primary effector of the augmentation of secretion caused by CT: (i) culture conditions were found in which CT caused large increases in secretion but very modest (or no) increases in cAMP; (ii) under other conditions, progressive increase in [CT] caused a maximum 2.5- to 3-fold increase in cAMP followed by a return to basal levels, whereas the secretory response saturated and remained stable; (iii) permeant cAMP analogs consistently enhanced secretion at low doses and inhibited at higher doses, but the peak enhancement was always much less than that achieved by an optimal dose of CT; (iv) the selective phosphodiesterase inhibitor Ro 20-1724 exhibited similar biphasic dose-response curves, the maximum enhancement again being small compared to that caused by CT itself. Both in vitro and in vivo, CT catalyzed transfer of ADP-ribose from NAD to two membrane proteins that comigrated on NaDodSO4/polyacrylamide gel electrophoresis with two CT substrates in other cell types, and these were identified by immunoblotting as Gs alpha. These results suggest that ADP-ribosylation of a cholera toxin substrate potentiates IgE-mediated secretion from RBL-2H3 cells by a largely cAMP-independent route.
Human genes for complement components C1r and C1s in a close tail-to-tail arrangement.
Authors: Kusumoto H , Hirosawa S , Salier J P , Hagen F S , Kurachi K
Published: May 2006

Complementary DNA clones for human C1s were isolated from cDNA libraries that were prepared with poly(A)+ RNAs of human liver and HepG2 cells. A clone with the largest cDNA insert of 2664 base pairs (bp) was analyzed for its complete nucleotide sequence. It contained 202 bp of a 5' untranslated region, 45 bp of coding for a signal peptide (15 amino acid residues), 2019 bp for complement component C1s zymogen (673 amino acid residues), 378 bp for a 3' untranslated region, a stop codon, and 17 bp of a poly(A) tail. The amino acid sequence of C1s was 40.5% identical to that of C1r, with excellent matches of tentative disulfide bond locations conserving the overall domain structure of C1r. DNA blotting and sequencing analyses of genomic DNA and of an isolated genomic DNA clone clearly showed that the human genes for C1r and C1s are closely located in a "tail-to-tail" arrangement at a distance of about 9.5 kilobases. Furthermore, RNA blot analyses showed that both C1r and C1s genes are primarily expressed in liver, whereas most other tissues expressed both C1r and C1s genes at much lower levels (less than 10% of that in liver). Multiple molecular sizes of specific mRNAs were observed in the RNA blot analyses for both C1r and C1s, indicating that alternative RNA processing(s), likely an alternative polyadenylylation, might take place for both genes.
Immunoglobulins from amyotrophic lateral sclerosis patients enhance spontaneous transmitter release from motor-nerve terminals.
Authors: Uchitel O D , Appel S H , Crawford F , Sczcupak L
Published: May 2006

Amyotrophic lateral sclerosis (ALS) is an incapacitating neuromuscular disease of unknown etiology. Although laboratory evidence is lacking, circumstantial evidence supports the importance of immune factors in the pathogenesis of ALS. In the present study immunoglobulins from 4 of 8 ALS patients induced a significant increase in spontaneous quantal transmitter release as monitored by miniature end-plate potential (MEPP) frequency in mouse phrenic nerve-diaphragm preparations at 23 degrees C, whereas immunoglobulins from normal individuals and from patients with other neuromuscular diseases had no effect. At 32 degrees C neither normal nor disease control immunoglobulins influenced MEPP frequency, but 8 of 11 ALS immunoglobulin samples produced a significant increase in spontaneous quantal transmitter release. The enhancing effect could be prevented by 10 mM Mg2+. No effects were noted on MEPP amplitude and muscle resting potential. These findings suggest that the presynaptic terminal of the motor neuron may be an early target and that immunological factors may play an important role in the disease process.
Isomeric prostaglandin F2 compounds arising from prostaglandin D2: a family of icosanoids produced in vivo in humans.
Authors: Wendelborn D F , Seibert K , Roberts L J
Published: May 2006

Prostaglandin (PG) D2 has been shown to be transformed by human 11-ketoreductase to 9 alpha,11 beta-PGF2, a biologically active metabolite that is produced in vivo. During the course of developing a mass spectrometric assay for 9 alpha,11 beta-PGF2, several compounds with characteristics similar to PGF2 were detected in both plasma and urine of normal humans by selected ion monitoring. Analysis of pooled plasma obtained from patients with mastocytosis during severe episodes of systemic mast cell activation associated with the release of markedly increased quantities of PGD2 was revealing in that all of these compounds were present in approximately 800-fold greater abundance compared to levels found in normal plasma, suggesting that these compounds arose from PGD2 metabolism. Complete electron impact mass spectra were obtained of these compounds in both plasma and urine; these spectra established that they were all isometric forms of PGF2. Approximately 16 isomeric PGF2 compounds were identified. Treatment with butylboronic acid indicated that the C-9 and C-11 hydroxyls were trans in approximately one-third of the compounds and cis in approximately two-thirds. Preliminary experiments suggest that PGD2 is a very labile compound in vivo and undergoes extensive isomerization, after which reduction by 11-ketoreductase yields a family of more stable isomeric PGF2 compounds. Elucidating the profile of biological activity of these compounds and their mechanism of formation will contribute importantly to our understanding of the biological consequences of PGD2 release in vivo. These results also bring into question the reliability of assays for PGF2 alpha and its metabolites in human biological fluids as a specific index of endogenous PGF2 alpha biosynthesis, as these assays may also measure in part isomeric PGF2 compounds arising from PGD2 metabolism.
Characterization of the cellular receptor for fibronectin through a hydropathic complementarity approach.
Authors: Brentani R R , Ribeiro S F , Potocnjak P , Pasqualini R , Lopes J D , Nakaie C R
Published: May 2006

It has been shown that a significant correlation is seen when the hydropathy scores of amino acids encoded by the coding strand of double-helical DNA are plotted against those of the noncoding strand. Thus, peptides encoded by complementary DNA strands might form amphiphilic structures and bind one another. We have used this approach to study the interaction between fibronectin (FN) and its cell receptor. Taking into consideration the nucleotide sequence from published rat cDNA clones that corresponds to the cell binding site (Arg-Gly-Asp-Ser) in the FN molecule, the deduced amino acid sequence found for the putative receptor binding site was Trp-Thr-Val-Pro-Thr-Ala. This peptide was chemically synthesized and coupled to an AH-Sepharose column. FN bound appreciably to this column and was eluted much more efficiently by a solution of Arg-Gly-Asp-Ser-containing peptide than by a solution of related but inactive Arg-Gly-Glu-Ser-containing peptide. Binding of labeled FN to receptor-rich MG63 human osteosarcoma cells was inhibited by the hexapeptide. The hexapeptide Gly-Ala-Val-Ser-Thr-Ala predicted similarly from the nucleotide sequence of human FN was equally efficient in such inhibition. Antibodies produced against Trp-Thr-Val-Pro-Thr-Ala recognized with equal efficiency Gly-Ala-Val-Ser-Thr-Ala in an ELISA assay. Furthermore, they were able to recognize a single 140-kDa band in whole-cell extracts from Chinese hamster ovary cells, attesting to their specificity. Identification of the recognized protein was provided by showing that this antibody was also able to bind to affinity-purified FN receptor from human osteosarcoma MG63 cells.
A 7-kDa region of the bacteriophage T7 gene 4 protein is required for primase but not for helicase activity.
Authors: Bernstein J A , Richardson C C
Published: May 2006

Bacteriophage T7 gene 4 protein, purified from phage-infected cells, consists of a mixture of 56- and 63-kDa species that provides helicase and primase activities required for T7 DNA replication. The 56-kDa species has been purified independently of the colinear 63-kDa species. Like a mixture of the two proteins, the 56-kDa protein binds single-stranded DNA in the presence of dTTP, catalyzes DNA-dependent hydrolysis of dTTP, and has helicase activity. In contrast to the mixture, the 56-kDa protein cannot catalyze template-dependent RNA primer synthesis. In the absence of a DNA template, both the 56-kDa protein and the mixture of the two species synthesize low levels of diribonucleotide. A putative "zinc finger" present near the amino terminus of the 63-kDa protein but absent from the 56-kDa protein may play a major role in the recognition of primase sites in the template.
Disulfide bonds and thermal stability in T4 lysozyme.
Authors: Wetzel R , Perry L J , Baase W A , Becktel W J
Published: May 2006

Disulfide bonds are thought to serve a stabilizing role in extracellular globular proteins, but little is known about the modes of stabilization or their mechanisms. Thermodynamic data presented here demonstrate that an engineered 3-97 disulfide bond previously shown to stabilize T4 lysozyme in vitro against irreversible thermal inactivation also stabilizes the molecule against reversible thermal unfolding. In this paper, we explore the relationship between the disulfide's thermodynamic contribution to protein folding and its role in providing resistance to irreversible thermal inactivation. In T4 lysozyme (C54V/C97S), a non-crosslinked mutant lacking the two cysteines found in the wild type, sensitivity toward irreversible thermal inactivation increases dramatically at temperatures above the melting temperature of the molecule. In addition, most of the lost activity can be restored by denaturation/renaturation with guanidine hydrochloride. In contrast, the crosslinked mutant T4 lysozyme (13C-97C/C54V) inactivates relatively slowly, even above its melting temperature, and the lost activity is not restored by denaturation/renaturation. These observations suggest that the predominant inactivation pathways for non-crosslinked T4 lysozymes are conformation related, while those for the crosslinked variant are insensitive to the conformational route and thus are susceptible only to slower processes of a chemical nature. We also show that multiple mutants, constructed to contain the 3-97 disulfide plus a temperature-sensitive lesion, are more stable than the wild type to irreversible inactivation even though they are less stable to reversible thermal unfolding. These findings together suggest that the 3-97 disulfide provides stability to irreversible inactivation primarily via a pathway that is independent of its thermodynamic contribution. The 3-97 disulfide may stabilize T4 lysozyme by restricting the unfolded state to a class of more compact structures with less exposed hydrophobic surface, compared to the unfolded states of non-crosslinked T4 lysozymes. The results have implications both for the use of the stabilizing potential of disulfide bonds in protein engineering and for their roles in protein function and evolution.
Neoplastic transformation of mouse mammary epithelial cells by in vitro exposure to N-methyl-N-nitrosourea.
Authors: Miyamoto S , Guzman R C , Osborn R C , Nandi S
Published: May 2006

High-efficiency neoplastic transformation of mouse mammary epithelial cells in primary collagen gel culture was induced by N-methyl-N-nitrosourea (MNU). Mammary epithelial cells, isolated from virgin BALB/c mice, were embedded within collagen gels and grown in a serum-free medium containing prolactin, progesterone, and linoleic acid. The cells were then treated with MNU on day 3 of culture and subsequently at weekly intervals for up to 4 weeks. Eleven to 14 days after the final carcinogen treatment, the cells were removed from the collagen gels and injected into the cleared mammary fat pads of syngeneic hosts to assay for transformed cell populations. A single exposure or multiple exposures of these cells to MNU was effective in inducing tumorigenic cells that produced palpable tumors as early as 6 weeks after transplantation. Two treatments with MNU (100 micrograms/ml) were optimal for neoplastic transformation and produced tumors in 79% of the injected fat pads. All the tumors originated at the site of injection and had extensive central necroses. Histological examination indicated that the tumors were mammary carcinomas. Secondary transplantation of tumor pieces into intact mammary glands produced palpable carcinomas of the same histology within 1-8 weeks. Control cells cultured for the same periods of time as MNU-treated cells produced only ductal outgrowths that were morphologically similar to those found in the mammary glands of adult virgin hosts. This system provides a distinct means to study the mechanism of mammary neoplastic transformation at cellular and molecular levels.
Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.
Authors: Baltus E , Hanocq-Quertier J , Hanocq F , Brachet J
Published: May 2006

The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the descendants of the injected blastomere. Cytological observations of the injected eggs show, in the arrested blastomeres, enlarged nuclei always surrounded by an intact nuclear envelope and containing uncondensed chromatin. The possible role of ras protein in meiosis and mitosis is discussed.
Two mitochondrial matrix proteases act sequentially in the processing of mammalian matrix enzymes.
Authors: Kalousek F , Hendrick J P , Rosenberg L E
Published: May 2006

The imported precursors of the mammalian matrix enzymes malate dehydrogenase [(S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] and ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) are cleaved to their mature subunits in two steps, each catalyzed by matrix-localized processing proteases. The number and properties of these proteases are the subjects of this report. We have identified and characterized two distinct protease activities in a crude matrix fraction from rat liver: processing protease I, which cleaves these precursors to the corresponding intermediate form; and processing protease II, which cleaves the intermediate forms to mature subunits. Protease I is insensitive to chelation by EDTA and to inactivation with N-ethylmaleimide; protease II is inhibited by 5 mM EDTA and is inactivated by treatment with N-ethylmaleimide. We have prepared from mitochondrial matrix an 800-fold-enriched protease I fraction free of protease II activity by using the following steps: ion exchange, hydroxyapatite, molecular sieving, and hydrophobic chromatography. Using similar procedures, we also have prepared an approximately 2000-fold-enriched protease II fraction, which has a trace amount of contaminating protease I. This enriched protease II fraction has little or no cleavage activity toward mitochondrial precursors but rapidly and efficiently converts intermediate forms to mature size. Finally, we show that protease I alone is sufficient to cleave the precursor of a third nuclear-encoded mitochondrial protein subunit--the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3]--to its mature size.
Targeting of an inducible toxic phenotype in animal cells.
Authors: Borrelli E , Heyman R , Hsi M , Evans R M
Published: May 2006

We have developed a toxic, or suicide, vector whose action is based on the targeted expression of the herpes simplex virus 1 thymidine kinase gene product in cultured cells or transgenic animals. This protein is able to convert nucleoside analogs such as acyclovir and 1-(2-deoxy-2-fluoro-beta-D-arabino-furanosyl)-5-iodouracil (FIAU) to toxic intermediates. The activation of these compounds disrupts cellular DNA replication, leading to rapid cell death. Neither acyclovir, FIAU, nor the herpes thymidine kinase alone is harmful to cells. This approach is simple and should have widespread applicability in studying lineage formation in cultured cells and transgenic animals.
Derived protein sequence, oligosaccharides, and membrane insertion of the 120-kDa lysosomal membrane glycoprotein (lgp120): identification of a highly conserved family of lysosomal membrane glycoproteins.
Authors: Howe C L , Granger B L , Hull M , Green S A , Gabel C A , Helenius A , Mellman I
Published: May 2006

The 120-kDa lysosomal membrane glycoprotein (lgp120) is an acidic, heavily glycosylated membrane protein enriched in the lysosomal membrane. To determine the basis for its selective transport to and stability in lysosomes, we have investigated the structure of lgp120. By using an oligonucleotide probe corresponding to the amino terminus of rat lgp120, we isolated and characterized cDNA clones containing the entire coding region. The deduced amino acid sequence demonstrates that lgp120 contains a putative signal peptide, 18 sites for N-linked glycosylation, a single membrane-spanning segment, and a short (11 amino acid) cytosolic tail. The sequence suggests a distinct domain organization, with two luminal glycosylated regions separated by a nonglycosylated proline-rich region. Proteolysis in detergent showed that the protein was not intrinsically resistant to exogenous or endogenous proteases. The N-linked oligosaccharides on lgp120, tetraantennary structures with two lactosamine repeats on one of the branches, were not different from those of glycoproteins on the plasma membrane. lgp120 was similar in its domain organization and portions of its amino acid sequence to the avian 100-kDa lysosomal membrane protein LEP100 [Fambrough, D. M., Takeyasu, K., Lippincott-Schwartz, J., Siegel, N. R. & Somerville, D. (1988) J. Cell Biol. 106, 61-67], and to a distinct 110-kDa lysosomal membrane protein (lgp110) that colocalizes with lgp120. The similarities between lysosomal membrane glycoproteins from diverse species, coupled with the fact that at least two distinct lysosomal membrane glycoproteins are expressed in a single species, indicate the existence of a conserved family of glycoproteins enriched in the lysosomal membrane.
An in vivo titration of regulatory factors required for expression of a fusion gene in transgenic sea urchin embryos.
Authors: Livant D L , Cutting A E , Britten R J , Davidson E H
Published: May 2006

We report that endogenous regulatory factors mediating expression of a lineage-specific sea urchin embryo gene can be titrated in vivo by introduction of a sufficient molar excess of DNA-binding sites. Thus we obtain an estimate of the quantity of limiting factor(s) required for developmental activation and transcriptional expression, which can be compared with estimates of factor prevalence obtained by measurements in vitro carried out under equilibrium conditions. A fusion construct in which the bacterial gene for chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol O3-acetyltransferase, EC 2.3.1.28) is controlled by cis-regulatory elements of the CyIIIa cytoskeletal actin gene (CyIIIa-CAT) was introduced in varying numbers of copies into sea urchin eggs. The activity of the CyIIIa-CAT fusion gene in 24-hr blastula-stage embryos was shown to saturate as the number of exogenous genes was increased. The mean number of CyIIIa-CAT fusion genes per nucleus at which half saturation was obtained was 105 +/- 40 (mean +/- SD). This result suggests that equilibrium parameters measured earlier in vitro may apply, at least approximately, within the embryo nuclei.
Monoclonal antibody identification of a 100-kDa membrane protein in HeLa cells and human spinal cord involved in poliovirus attachment.
Authors: Shepley M P , Sherry B , Weiner H L
Published: May 2006

Unique receptor sites for poliovirus are considered to be the primary determinant of the virus' cell and tissue-type specificity. To study the poliovirus-cell interaction, eight monoclonal antibodies that specifically block the cytopathic effects of poliovirus were generated by using HeLa cell preparations as immunogen and a newly developed colorimetric screening assay. Plaque-inhibition assays confirmed the viral specificity of the antibodies, and when one antibody, AF3, was used as a probe in immunoblots of cell membrane preparations, it detected a 100-kDa band in only those cell lines and tissues permissive for poliovirus infection. AF3 also specifically inhibited radiolabeled poliovirus binding to cells. In terms of tissue specificity, AF3 detected the 100-kDa band in membrane preparations from human spinal cord but not in organ homogenates of human kidney or in murine tissue, including the central nervous system. Furthermore, AF3 detected the band in a human-mouse hybrid cell line containing human chromosome 19, which confers permissivity for poliovirus infection, but the antibody did not detect the band in a human chromosome 19-deficient subclone. In an immunohistochemical study of the human brainstem, AF3 stained neurons in the reticular formation and clusters of brainstem neurons, consistent with the known pattern of damage caused by poliovirus infection in the brainstem. Furthermore, AF3 reacted with human peripheral mononuclear cells, consistent with the known replication of poliovirus in Peyer's patches and tonsils. These results strongly suggest that the 100-kDa band detected by antibody AF3 is, or is closely associated with, the poliovirus receptor site.
Direct measurement of nitroxide pharmacokinetics in isolated hearts situated in a low-frequency electron spin resonance spectrometer: implications for spin trapping and in vivo oxymetry.
Authors: Rosen G M , Halpern H J , Brunsting L A , Spencer D P , Strauss K E , Bowman M K , Wechsler A S
Published: May 2006

The pharmacokinetics of two nitroxides were investigated in isolated rat hearts situated in a low-frequency electron spin resonance spectrometer. The spin labels 2,2,3,3,5,5-hexamethyl-1-pyrrolidinyloxy and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy were chosen for their physiochemical analogy to the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and its corresponding spin-trapped adduct, 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxy (DMPO-OH). The bioreductive rates of the two nitroxides were measured during constant perfusion as well as during ischemia and are discussed in terms of a two-compartment pharmacokinetic model. These data provide information necessary to the design and application of spin traps to detect oxy radicals during reperfusion of ischemic tissue and suggest the feasibility of monitoring free-radical processes in intact, functioning mammalian tissues by using a low-frequency electron spin resonance spectrometer.
Hypertension induces tissue-specific gene suppression of a fatty acid binding protein in rat aorta.
Authors: Sarzani R , Claffey K P , Chobanian A V , Brecher P
Published: May 2006

The effect of hypertension on the expression of a fatty acid binding protein localized in the rat aorta was studied. The presence of rat heart fatty acid binding protein (hFABP) was documented in aortic tissue by using a cDNA probe and polyclonal antibodies. Hypertension was induced in groups of rats by implantation of deoxycorticosterone acetate in conjunction with 1% salt in the drinking water (deoxycorticosterone/salt). By the third week of this treatment a marked reduction (by a factor of 20) in the expression of hFABP mRNA in aorta was found, concomitant with a reduction in immunologically detectable protein, suggesting transcriptional regulation. This effect was tissue specific, since no change in the normal amounts of hFABP mRNA in heart, skeletal muscle, or kidney was found. This reduction in aortic hFABP mRNA was also found in mildly hypertensive uninephrectomized rats given salt but no deoxycorticosterone and in normotensive rats given deoxycorticosterone but no excess salt intake. A marked decrease in aortic hFABP mRNA also was observed in the Goldblatt two kidney-one clip hypertensive model, and administration of angiotensin II for 6 days by osmotic minipump also caused a reduction. These findings suggest that hFABP is under complex regulation in aortic tissue and is suppressed by arterial hypertension.
Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport.
Authors: Block M R , Glick B S , Wilcox C A , Wieland F T , Rothman J E
Published: May 2006

N-Ethylmaleimide (NEM) inhibits protein transport between successive compartments of the Golgi stack in a cell-free system. After inactivation of the Golgi membranes by NEM, transport can be rescued by adding back an appropriately prepared cytosol fraction. This complementation assay has allowed us to purify the NEM-sensitive factor, which we term NSF. The NEM-sensitive factor is a tetramer of 76-kDa subunits, and appears to act catalytically, one tetramer leading to the metabolism of numerous transport vesicles.
Cleavage of the cII protein of phage lambda by purified HflA protease: control of the switch between lysis and lysogeny.
Authors: Cheng H H , Muhlrad P J , Hoyt M A , Echols H
Published: May 2006

The activity of the cII protein of phage lambda is probably the critical controlling factor in the choice of the lytic or lysogenic pathway by an infecting virus. Previous work has established that cII activity is regulated through the turnover of cII protein; the products of the hflA and hflB loci of Escherichia coli are needed for a degradative reaction, and lambda cIII functions in stabilizing cII. By using the cloned hflA locus, we have purified a cII-cleaving enzyme that we term HflA. Purified HflA contains three polypeptides; at least two of the subunits are products of the hflA region, and the third is probably a cleavage product of the larger of these two hflA-encoded polypeptides. The HflA protease activity cleaves cII to small fragments. We conclude that the switch between lambda developmental pathways involves regulated cleavage of cII by the specific protease HflA.
Holliday junctions in FLP recombination: resolution by step-arrest mutants of FLP protein.
Authors: Jayaram M , Crain K L , Parsons R L , Harshey R M
Published: May 2006

The FLP "recombinase" of the 2-micron circle yeast plasmid can resolve synthetic FLP site-Holliday junctions. Mutants of the FLP protein that are blocked in recombination but are normal in substrate cleavage can also mediate resolution. The products of resolution by these mutants are almost exclusively nicked molecules with a protein-bound 3' end. There is no significant asymmetry in strand cleavage (top versus bottom) by the mutants in linear or in circular FLP substrates; nor is there a bias in resolution (toward parentals or toward recombinants) of Holliday junctions (corresponding to top- or to bottom-strand exchange) by wild-type FLP. During normal FLP recombination, a small amount of the expected Holliday intermediate can be detected.
DNA polymerase of bacteriophage T4 is an autogenous translational repressor.
Authors: Andrake M , Guild N , Hsu T , Gold L , Tuerk C , Karam J
Published: May 2006

In bacteriophage T4 the protein product of gene 43 (gp43) is a multifunctional DNA polymerase that is essential for replication of the phage genome. The protein harbors DNA-binding, deoxyribonucleotide-binding, DNA-synthesizing (polymerase) and 3'-exonucleolytic (editing) activities as well as a capacity to interact with several other T4-induced replication enzymes. In addition, the T4 gp43 is a repressor of its own synthesis in vivo. We show here that this protein is an autogenous repressor of translation, and we have localized its RNA-binding sequence (translational operator) to the translation initiation domain of gene 43 mRNA. This mechanism for regulation of T4 DNA polymerase expression underscores the ubiquity of translational repression in the control of T4 DNA replication. Many T4 DNA polymerase accessory proteins and nucleotide biosynthesis enzymes are regulated by the phage-induced translational repressor regA, while the T4 single-stranded DNA-binding protein (T4 gp32) is, like gp43, autogenously regulated at the translational level.
Replacement of insulin receptor tyrosine residues 1162 and 1163 does not alter the mitogenic effect of the hormone.
Authors: Debant A , Clauser E , Ponzio G , Filloux C , Auzan C , Contreres J O , Rossi B
Published: May 2006

Chinese hamster ovary transfectants that express insulin receptors in which tyrosine residues 1162 and 1163 were replaced by phenylalanine exhibit a total inhibition of the insulin-mediated tyrosine kinase activity toward exogenous substrates [histone, casein, and poly(Glu/Tyr)]; this latter activity is associated with total inhibition of the hypersensitivity reported for insulin in promoting 2-deoxyglucose uptake. We now present evidence that the twin tyrosines also control the insulin-mediated stimulation of glycogen synthesis. Surprisingly, this type of Chinese hamster ovary transfectant is as hypersensitive to insulin for its mitogenic effect as are Chinese hamster ovary cells expressing many intact insulin receptors. Such data suggest that (i) the insulin mitogenic effect routes through a different pathway than insulin uses to activate the transport and metabolism of glucose and (ii) the mitogenic effect of insulin is not controlled by the twin tyrosines. At the molecular level, the solubilized mutated receptor has no insulin-dependent tyrosine kinase activity, whereas this receptor displays measurable insulin-stimulated phosphorylation of its beta subunit in 32P-labeled cells. We therefore propose that the autocatalytic phosphorylating activity of the receptor reports a cryptic tyrosine kinase activity that cannot be visualized by the use of classical exogenous substrates.
Identification and subcellular localization of proteins that are rapidly phosphorylated in tyrosine in response to colony-stimulating factor 1.
Authors: Sengupta A , Liu W K , Yeung Y G , Yeung D C , Frackelton A R , Stanley E R
Published: May 2006

To investigate growth factor-mediated signal transduction, we have studied phosphorylation events that take place within seconds of the binding of colony-stimulating factor 1 (CSF-1) to its cell-surface receptor. CSF-1 stimulated rapid tyrosine phosphorylation of cellular proteins in murine BAC1.2F5 macrophages at 37 degrees C and 4 degrees C. The pattern of CSF-1-stimulated tyrosine phosphorylation of at least 15 different proteins at both temperatures was similar and unchanged by treatment of the lysate with reducing agent. With the exception of the 185-kDa CSF-1 receptor, a 260-kDa protein and a 133-kDa protein, the proteins were predominantly cytoplasmic. At 37 degrees C, all the proteins were phosphorylated within 30 sec of addition of growth factor. At 4 degrees C, CSF-1 receptor sites were saturated after 2 min of incubation in the presence of high concentrations of CSF-1 and differences in the order of appearance of phosphorylated proteins were observed: 185 kDa (CSF-1 receptor) (by 2 min); 99 kDa (by 4 min); 125 kDa (by 10 min); 61 kDa (by 30 min); and 260 kDa, 84 kDa, and 41 kDa (by 180 min). In addition to stimulating the phosphorylation of these proteins in tyrosine, CSF-1 caused dephosphorylation of phosphorylated serine residues on the receptor. As neither CSF-1 nor its receptor is internalized at 4 degrees C, analysis of these early reactions and the phosphotyrosine-containing proteins in intact cells under these conditions should lead to an understanding of the early events in growth factor receptor-mediated signal transduction.
Peptide-mediated modulation of T-cell allorecognition.
Authors: Eckels D D , Gorski J , Rothbard J , Lamb J R
Published: May 2006

Antigen-specific helper T cells recognize a complex of peptide antigen and class II major histocompatibility complex (MHC) gene products. Whether T cells recognize MHC class II alloantigen by a similar mechanism or the native conformation of MHC molecules themselves has yet to be determined. The demonstration that peptide antigens bind directly and specifically to class II molecules has allowed us to examine the influence of foreign peptide binding on T-cell recognition of allogeneic MHC molecules. We report here that an immunodominant, HLA-DR1-restricted peptide of influenza virus hemagglutinin (HA residues 306-320) is able to modulate the recognition of alloantigen by human DR1-specific T-cell clones. For some T-cell clones, but not all, the HA peptide inhibited allorecognition in a dose-dependent manner. However, in one instance, the proliferative response to alloantigen was enhanced in the presence of HA peptide. These results suggest that the specificities of T-cell responses to allogeneic MHC molecules are heterogeneous, which may be influenced by different peptides occupying the class II MHC combining site and by the diversity of antigen-specific receptors of T lymphocytes recognizing the same MHC/peptide complex.
Physical linkage of a human immunoglobulin heavy chain variable region gene segment to diversity and joining region elements.
Authors: Schroeder H W , Walter M A , Hofker M H , Ebens A , Willems van Dijk K , Liao L C , Cox D W , Milner E C , Perlmutter R M
Published: May 2006

Antibody genes are assembled from a series of germ-line gene segments that are juxtaposed during the maturation of B lymphocytes. Although diversification of the adult antibody repertoire results in large part from the combinatorial joining of these gene segments, a restricted set of antibody heavy chain variable (VH), diversity (DH), and joining (JH) region gene segments appears preferentially in the human fetal repertoire. We report here that one of these early-expressed VH elements (termed VH6) is the most 3' VH gene segment, positioned 77 kilobases on the 5' side of the JH locus and immediately adjacent to a set of previously described DH sequences. In addition to providing a physical map linking human VH, DH, and JH elements, these results support the view that the programmed development of the antibody VH repertoire is determined in part by the chromosomal position of these gene segments.
Molecular dynamics in the membranes of helper T cells.
Authors: Kupfer A , Singer S J
Published: May 2006

We provide evidence that redistributions and interactions of integral proteins in the fluid membranes of helper T (Th) cells may play important roles in Th-cell activation. A particular monoclonal antibody, 3D3, directed to a clonotypic determinant on the T-cell receptor (TCR) of the cloned Th-cell line D10, had previously been shown to be distinctively capable of directly activating D10 cells at low concentrations. We demonstrate here by immunofluorescence experiments that it is also distinctively able itself to produce a clustering (capping) of the TCRs on the D10 cell surface. Simultaneously, by means of double-immunofluorescence experiments, we find that the 3D3-induced clustering of the TCRs distinctively produces a co-clustering of the accessory molecule CD4 with the TCR clusters, although the CD4 and TCR molecules are normally independent of one another in the D10 cell membrane. These results, and related ones previously obtained from studies of the interactions of D10 Th cells with antigen-presenting cells, are analyzed to suggest that the membrane clustering of TCRs and the induced TCR-CD4 interactions are critical to the signaling events in Th-cell activation.
Physics and mathematics at the frontier
Authors: Gross David J.
Published: May 2006

The relation between elementary particle physics and mathematics is explored. The beauty and effectiveness of the mathematical structures that appear in fundamental physics are discussed.
A genomic DNA segment from Petunia hybrida leads to increased transformation frequencies and simple integration patterns.
Authors: Meyer P , Kartzke S , Niedenhof I , Heidmann I , Bussmann K , Saedler H
Published: May 2006

A 2-kilobase (kb) genomic fragment was selected from Petunia hybrida that increased transformation efficiencies by at least a factor of 20 after direct DNA transfer to petunia and tobacco protoplasts when supercoiled plasmid DNA was used. Because of this effect this fragment was named transformation booster sequence (TBS). Increased transformation frequencies were observed for plasmids that contained either the 2-kb fragment in dimeric or monomeric form or an internal 1.1-kb fragment of TBS. Analysis of transformants revealed that preferentially one copy of foreign DNA is integrated. Thus, TBS improves the poor transformation frequencies of direct gene transfer using circular plasmids, while it conserves the simple integration pattern that is important for practical applications. Possible mechanisms of TBS action are discussed.
CD45 regulates signal transduction and lymphocyte activation by specific association with receptor molecules on T or B cells.
Authors: Ledbetter J A , Tonks N K , Fischer E H , Clark E A
Published: May 2006

Evidence is presented that the leukocyte common antigen CD45 can regulate both signal transduction by lymphocyte receptor molecules and T- and B-cell proliferation in a manner dependent on specific interactions between these receptors on the cell surface. Formation of homoaggregates of CD3, CD2, or CD28 on the surface of T cells induced by crosslinking with monoclonal antibodies (mAbs) results in an increase in cytoplasmic free calcium concentration ([Ca2+]i). This increase in [Ca2+]i was abolished when these receptors were crosslinked to CD45 on the cell surface. In contrast, the increase in [Ca2+]i induced by formation of homoaggregates of CD4 was strongly amplified when CD4 was coupled to CD45. T-cell proliferation initiated by immobilized anti-CD3 was inhibited by anti-CD45 or anti-CD45R when immobilized on the same surface, but not when in solution. Similarly, proliferation after stimulation of the CD2 and CD28 receptors was inhibited when a CD45 mAb was crosslinked to either CD2 or CD28 mAbs, but not when a CD45-specific mAb was bound to the cell surface separately. In B cells, the increase in [Ca2+]i and resulting proliferation induced by crosslinking either the CD19 or Bgp95 receptors was inhibited by coupling these molecules to CD45. Thus, CD45 appears to modify other cellular receptors functionally when brought into close physical association with them. The homology of the CD45 conserved cytoplasmic domains with a major human placental protein tyrosine phosphatase suggests that the effects of CD45 described here result from alterations in the phosphorylation state of tyrosyl residues in membrane-associated proteins.
Interleukin 2-dependent and interleukin 2-independent pathways of regulation of thymocyte function by interleukin 6.
Authors: Le J M , Fredrickson G , Reis L F , Diamantstein T , Hirano T , Kishimoto T , Vilcek J
Published: May 2006

Recombinant human interleukin 6 (IL-6), also termed B-cell-stimulatory factor 2 (BSF-2) or interferon-beta 2, was found to stimulate the proliferation of mouse thymocytes costimulated with phytohemagglutinin (PHA). In addition, IL-6 synergistically enhanced the stimulation of thymocyte proliferation by recombinant human interleukin 1 (IL-1) or interleukin 2 (IL-2). Mature thymocytes lacking peanut agglutinin receptor are the main target of IL-6 action. Incubation of thymocytes with IL-6 in the presence of PHA resulted in an increased expression of the IL-2 receptor (IL-2R) as demonstrated by flow cytometry. Monoclonal antibody specific for the p55 chain of the murine IL-2R significantly reduced IL-6-stimulated thymocyte proliferation in the presence of the optimal concentration of PHA. However, the same monoclonal antibody failed to reduce IL-6-driven thymocyte proliferation in the presence of a suboptimal PHA concentration, suggesting that IL-6 stimulates thymocyte proliferation by way of IL-2-dependent and IL-2-independent pathways. These results indicate that, in addition to its earlier demonstrated ability to promote B-cell differentiation and growth, IL-6 also acts as a growth regulator in cells of the T-lymphocyte lineage. IL-6 is emerging as an important regulatory cytokine with multiple actions on immune functions.
Site of covalent attachment of alpha-scorpion toxin derivatives in domain I of the sodium channel alpha subunit.
Authors: Tejedor F J , Catterall W A
Published: May 2006

Purified and reconstituted sodium channels from rat brain have been photoaffinity labeled with a photoactivable derivative of the alpha-scorpion toxin V from Leiurus quinquestriatus (LqTx). A battery of sequence-specific antibodies has been used to determine which of the peptides produced by chemical and enzymatic cleavage of the photolabeled sodium-channel alpha subunit contain covalently attached LqTx. Nearly all of the covalently attached LqTx is found within homologous domain I. Two site-directed antisera, which recognize residues 317 to 335 and residues 382 to 400, respectively, specifically immunoprecipitate a 14-kDa peptide produced by CNBr digestion to which LqTx is covalently attached. It is proposed that a portion of the receptor site for alpha-scorpion toxins is formed by peptide segment(s) between amino acid residues 335 and 378 which is located in an extracellular loop between transmembrane helices S5 and S6 of homologous domain I of the sodium channel alpha subunit.