bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple signaling pathways control stimulus-secretion coupling in rat peritoneal mast cells.
Authors: Penner R
Published: May 2006

Fura-2 and membrane capacitance measurements were performed to investigate intracellular Ca2+ concentration [( Ca2+]i) and secretory responses of rat peritoneal mast cells following secretagogue stimulation. Compound 48/80 and internally applied guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) induced transient rises in [Ca2+]i and caused membrane capacitance increases as secretion occurred. The 48/80-induced Ca2+ transients and secretory responses were blocked by guanosine 5'-[beta-thio]diphosphate and neomycin, indicating that inositolphospholipid breakdown mediated by guanine nucleotide-binding regulatory protein (G protein) plays an important role in stimulus-secretion coupling. However, pertussis toxin did not block Ca2+ transients induced by 48/80 or GTP[gamma-S], whereas secretory responses were either abolished (48/80) or developed only after a considerable delay (GTP[gamma-S]). Similar effects were obtained by perfusing cells with cAMP: (i) Ca2+ transients following stimulation with 48/80 remained unaffected by cAMP, but secretory responses were abolished; (ii) GTP[gamma-S] induced normal Ca2+ transients and degranulation in the presence of cAMP. Pretreatment of mast cells with phorbol 12-myristate 13-acetate (PMA) abolished 48/80- and GTP[gamma-S]-induced Ca2+ transients (but not inositol trisphosphate-induced Ca2+ transients), whereas secretion still occurred. At the same time, the Ca2+ requirement for secretion was reduced by PMA. These results indicate that secretion in mast cells is under control of an as yet unidentified signaling pathway that involves a G protein. This pathway is distinct from inositolphospholipid turnover and may provide the triggering mechanism for secretion, whereas the inositolphospholipid pathway serves to increase [Ca2+]i and renders the secretory process more sensitive to [Ca2+]i by activating protein kinase C. Persistent activation of protein kinase C through phorbol ester imposes negative feedback control on the inositolphospholipid pathway, whereas cAMP may inhibit the unidentified signaling pathway.
Elevated expression of thymidylate synthase cycle genes in cisplatin-resistant human ovarian carcinoma A2780 cells.
Authors: Scanlon K J , Kashani-Sabet M
Published: May 2006

Activity of the thymidylate synthase cycle was compared in the human ovarian carcinoma cell line A2780 and a subline that is resistant to cisplatin by a factor of 3. Resistant cells exhibited a 3-fold increase in mRNA for both dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+-oxidoreductase, EC 1.5.1.3) and thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) when compared with the parent line. Resistance to cisplatin also resulted in a 2.5-fold increase in enzyme activity for dihydrofolate reductase and thymidylate synthase; however, this increase did not result from amplification of the genes for these two enzymes. These data suggest that the initial step of cisplatin resistance in A2780 cells is a consequence of enhanced expression of the thymidylate synthase cycle.
Several “pathogenesis-related” proteins in potato are 1,3-β-glucanases and chitinases
Authors: Kombrink Erich , Schröder Martin , Hahlbrock Klaus
Published: May 2006

Chitinase {poly[1,4-(
N
-acetyl-β-D-glucosaminide)]glycanohydrolase, EC 3.2.1.14} and 1,3-β-glucanase (1,3-β-D-glucan 3-glucanohydrolase, EC 3.2.1.6) activities increased rapidly in potato (
Solanum tuberosum
) leaves inoculated with the pathogenic fungus
Phytophthora infestans
or treated with fungal elicitor. The enzyme activities were resolved into a total of two distinct 1,3-β-glucanases and six proteins with chitinase activity. By several criteria, all of these proteins are classified as “pathogenesis-related” proteins whose biochemical functions have so far been unknown. Some of them constitute a major portion of the proteins accumulating in the intercellular space of infected potato leaves and are assumed to play an important role in pathogen defense.
Evidence for a physiological role of hypothalamic gastrin-releasing peptide to suppress growth hormone and prolactin release in the rat.
Authors: Kentroti S , Dees W L , McCann S M
Published: May 2006

Gastrin-releasing peptide (GRP) is localized to hypothalamic neurons and is a potent inhibitor of basal and growth hormone (GH)-releasing factor-induced GH secretion in the rat. It also acts similarly to inhibit opiate- and stress-induced prolactin (PRL) release. To determine the physiological significance of the peptide in the control of the release of these two hormones, a highly specific antiserum against GRP was injected into the third brain ventricle to immunoneutralize hypothalamic GRP. The injection of the antiserum initially did not alter levels of the hormones; however, both PRL and GH levels in the plasma began to increase within 3 and 3.5 hr, respectively. They were still significantly elevated 24 hr after the injection. There was no change in the plasma levels of either hormone in animals injected intraventricularly with a similar volume of normal rabbit serum (NRS). Mean plasma GH levels 24 hr after antiserum injection were more than twice those of the NRS-injected controls, whereas the PRL concentrations were 14-fold higher in the antiserum injected as compared to the control NRS-injected animals. A second similar injection of antiserum 24 hr after the first administration resulted in a slight and transient further increase in both GH and PRL levels so that they were both significantly (P less than 0.001) higher than those of the animals given a second injection of NRS. The anti-GRP antiserum was highly specific for GRP by radioimmunoassay procedures and this antiserum produced positive immunostaining of GRP neuronal perikarya and terminals within discrete hypothalamic nuclei. Beaded fibers and terminals were observed in the suprachiasmatic nucleus (SCN) and the area lateral and dorsal to the SCN in the region of the periventricular nucleus (PeVN). GRP-positive perikarya were observed in the parvocellular neurons of the paraventricular nucleus. In addition, GRP-positive cell bodies were observed in the PeVN in close proximity to the third ventricle. Furthermore, the median eminence displayed no immunostaining for GRP, and all traces of positive staining were abolished by preabsorption of the antiserum with GRP-27 (30 micrograms/ml), confirming the specificity of the antiserum. The combined results with immunoneutralization of GRP and the immunostaining of GRP neuronal elements in the hypothalamus support the physiological role of this peptide in the inhibitory control of both GH and PRL release.
Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes.
Authors: Zhang H , Wang J C , Liu L F
Published: May 2006

Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role.
Transposable and nontransposable elements similar to the I factor involved in inducer-reactive (IR) hybrid dysgenesis in Drosophila melanogaster coexist in various Drosophila species.
Authors: Simonelig M , Bazin C , Pelisson A , Bucheton A
Published: May 2006

The I factor is a transposable element controlling inducer-reactive (IR) hybrid dysgenesis in Drosophila melanogaster, which occurs when males from the class of inducer strains are crossed with females from the class of reactive strains. Inducer strains contain several copies of the complete 5.4-kilobase (kb) I factor at various sites on the chromosomal arms; reactive strains contain no complete I factor. Incomplete and defective I elements occur at constant locations in pericentromeric heterochromatin of both types of strains. The 5.4-kb I factors transpose, whereas incomplete I elements do not transpose. The constant location of defective I elements in all strains indicates that they were in the genome before the spread of D. melanogaster throughout the world. Sequences homologous to I occur in other Drosophila species, and their distribution correlates with the phylogenetic relationships between species. We have studied the organization of I homologues in Drosophila simulans and Drosophila teissieri. These species seem to contain both transposable I elements, even though their structure may differ from that of the 5.4-kb I factors of the inducer strains of D. melanogaster, and nontransposable I elements, which are always at the same place in the genome when different stocks of the same species are compared. These results suggest that both mobile and nonmobile I elements are very old components of the Drosophilidae genome.
Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants.
Authors: Hizi A , McGill C , Hughes S H
Published: May 2006

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional amino-terminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptase-related protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies.
Synthetic peptides corresponding to protective epitopes of Escherichia coli digalactoside-binding pilin prevent infection in a murine pyelonephritis model.
Authors: Schmidt M A , O'Hanley P , Lark D , Schoolnik G K
Published: May 2006

Synthetic peptides corresponding to five segments of a globoside (Gal-Gal)-binding pilin sequence [residues 5-12 (R5-12), R65-75, R93-104, R103-116, and R131-143], cyanogen bromide fragment II (CNBr-II, R53-163), and purified, intact Gal-Gal pili were prepared as vaccines and tested for their efficacy in a BALB/c murine model of pyelonephritis. Intact Gal-Gal pili, CNBr-II, and synthetic peptides R5-12 and R65-75 engendered antibodies that bound the homologous pilin protein and prevented urine and renal colonization in most vaccine recipients. Protection correlated with serum anti-pilus IgG ELISA titers greater than or equal to 1:250. The efficacy afforded by synthetic peptides R5-12 and R65-75 in vaccinated mice indicates that linear "antigenic" determinants in separate cyanogen bromide fragments encode "protective" epitopes. Peptides R93-104, R103-116, and R131-143 lacked efficacy, indicating that not all regions of the sequence are serologically equivalent. The crossreactivity of the peptide antisera for different Gal-Gal pilins was also assessed and correlated with the sequence homology of the corresponding regions. Antiserum to peptide R65-75, which corresponds to a region of unconserved sequence in heterologous pilins, bound only the homologous pilin. Thus, it specifies a type-specific protective epitope. Antiserum to synthetic peptide R5-12, which corresponds to a region of conserved sequence, bound Gal-Gal pilins from seven of eight pyelonephritis strains, indicating that it specifies a crossreacting protective epitope.
Rod light adaptation may be mediated by acceleration of the phosphodiesterase-guanylate cyclase cycle.
Authors: Kondo H , Miller W H
Published: May 2006

We compare the retinal rod photocurrent before and after introduction of an hydrolysis-resistant analog of GTP into the outer segment by the whole-cell patch technique. Others have shown that GTP bound to transducin leads to the hydrolysis of cyclic GMP, causing the response to light--a decrease in dark current. The hydrolysis-resistant GTP analog prolongs the response to a bright flash, which leads us to suggest that prolonged transducin activation by bright light desensitizes the rod by a prolonged decrease in dark current. Recovery from the response to a bright flash does occur after introduction of the analog; that recovery requires acceleration of cyclase activity rather than inhibition of phosphodiesterase. The analog mimics light adaptation by desensitizing the rod and speeding the recovery from a dim flash. The analog plus light or light adaptation prolongs the activities of transducin and phosphodiesterase (oligonucleate 5'-nucleotidohydrolase, EC 3.1.4.1) to mediate desensitization by reducing the dark current. Hence, this faster recovery from a dim flash would be by increased activity of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] rather than by inhibited phosphodiesterase. Accelerated activity of guanylate cyclase may speed recovery by response truncation. We conclude that transducin, activated by photolyzed rhodopsin, may lead to increased activity of both phosphodiesterase and guanylate cyclase to mediate the desensitization and the faster recovery of the light-adapted response.
Surface coil localization of 31P NMR signals from orthotopic human kidney and liver.
Authors: Jue T , Rothman D L , Lohman J A , Hughes E W , Hanstock C C , Shulman R G
Published: May 2006

By incorporating the hyperbolic secant inversion pulses with the image-selected in vivo spectroscopy localization technique and by applying a gradient-echo imaging method, we have selected only the 31P NMR signals from orthotopic human kidney and liver, using a single concentric 1H/31P surface coil. Corresponding to the experimental results on animal studies, the phosphocreatine signal is dramatically reduced in the localized spectra. Our localization strategy also allows us to shim easily on the well-defined volume of interest and leads to high-resolution spectra that exhibit multiplet structure. Our results indicate that we can obtain localized signals from deep small organs and point the way for other human metabolism studies.
Restoration of phototrophic growth in a mutant of Chlamydomonas reinhardtii in which the chloroplast atpB gene of the ATP synthase has a deletion: an example of mitochondria-dependent photosynthesis.
Authors: Lemaire C , Wollman F A , Bennoun P
Published: May 2006

The Chlamydomonas reinhardtii mutant FUD50 has a deletion in the atpB gene of the chloroplast ATP synthase [Woessner, J. P., Masson, A., Harris, E. H., Bennoun, P., Gillham, N. W., and Boynton, J. E. (1984) Plant Mol. Biol. 3, 177-190]. We have isolated a suppressed strain (FUD50su) that can grow under phototrophic conditions, although it still showed no synthesis of the beta subunit of coupling factor 1. Thylakoid membranes of the FUD50su strain were similar to those of the original FUD50 strain, in that they both lacked all the subunits making up the chloroplast ATP synthase complex. We show that photosynthesis in FUD50su is sensitive to inhibitors such as antimycin, specific for mitochondrial electron transport. This observation indicates that photosynthesis in the FUD50su strain is achieved through an unusual interaction between mitochondria and chloroplast. Exportation of light-induced reduced compounds from the chloroplast to the mitochondria elicits ATP formation in the latter, and ATP is subsequently imported to the chloroplast. The activation of such an ATP shuttle coupled to an NADPH shuttle would thus provide the reducing power and the free energy needed for carbon assimilation in a chloroplast that lacks chloroplast ATP synthase.
Detection of human muscle glycogen by natural abundance 13C NMR.
Authors: Avison M J , Rothman D L , Nadel E , Shulman R G
Published: May 2006

Natural abundance 13C nuclear magnetic resonance spectroscopy was used to detect signals from glycogen in the human gastrocnemius muscle. The reproducibility of the measurement was demonstrated, and the ability to detect dynamic changes was confirmed by measuring a decrease in muscle glycogen levels after exercise and its subsequent repletion. Single frequency gated 1H decoupling was used to obtain decoupled natural abundance 13C NMR spectra of the C-1 position of muscle glycogen.
Three binding sites for AraC protein are required for autoregulation of araC in Escherichia coli.
Authors: Hamilton E P , Lee N
Published: May 2006

Three binding sites for AraC protein were shown to be required for the autoregulation of araC: araI1, araO1, and araO2. Selective inactivation of AraC-binding sites on the DNA demonstrated that araO1 and araO2 are required in vivo to produce repression of araC in the presence of arabinose, whereas araI1 and araO2 are required in its absence. We found that the low-affinity site araO2 is essential for araC autoregulation; araO1 and araI1 provide high-affinity AraC-binding sites, which allow cooperative binding at araO2. Profound effects on the araBAD promoter and the araC promoter are produced by ligand-induced changes in AraC occupancy of functional sites on the DNA. We suggest that AraC exerts its multiplicity of controls through two alternative states of cooperative interactions with DNA and we illustrate this with a model. This model presents our interpretations of activation and repression of the araBAD operon and the autoregulation of the araC gene.
The cytolytic C5b-9 complement complex: feedback inhibition of complement activation.
Authors: Bhakdi S , Maillet F , Muhly M , Kazatchkine M D
Published: May 2006

We describe a regulatory function of the terminal cytolytic C5b-9 complex [C5b-9(m)] of human complement. Purified C5b-9(m) complexes isolated from target membranes, whether in solution or bound to liposomes, inhibited lysis of sensitized sheep erythrocytes by whole human serum in a dose-dependent manner. C9 was not required for the inhibitory function since C5b-7 and C5b-8 complexes isolated from membranes were also effective. No effect was found with the cytolytically inactive, fluid-phase SC5b-9 complex. However, tryptic modification of SC5b-9 conferred an inhibitory capacity to the complex, due probably to partial removal of the S protein. Experiments using purified components demonstrated that C5b-9(m) exerts a regulatory effect on the formation of the classical- and alternative-pathway C3 convertases and on the utilization of C5 by cell-bound C5 convertases. C5b-9(m) complexes were unable to inhibit the lysis of cells bearing C5b-7(m) by C8 and C9. Addition of C5b-9(m) to whole human serum abolished its bactericidal effect on the serum-sensitive Escherichia coli K-12 strain W 3110 and suppressed its hemolytic function on antibody-sensitized, autologous erythrocytes. Feedback inhibition by C5b-9(m) represents a biologically relevant mechanism through which complement may autoregulate its effector functions.
Continuous synthesis of two protein-kinase-C-related proteins after down-regulation by phorbol esters.
Authors: Borner C , Eppenberger U , Wyss R , Fabbro D
Published: May 2006

The phorbol 12-myristate 13-acetate (PMA)-dependent down-regulation of immunoprecipitable protein kinase C was studied in human breast cancer cell lines that display different growth inhibitions toward the tumor promoter. PMA induces translocation of [35S]methionine-prelabeled cytosolic protein kinase C to membranes, followed by complete degradation of the enzyme (t1/2, 2 hr). PMA does not affect the protein kinase C synthesis; 20-80% of total protein kinase C of control cells was still immunoprecipitable as membrane-bound 74- and 80-kDa protein kinase C-related polypeptides if cells were allowed to incorporate [35S]methionine during PMA exposure for greater than 6 hr. These two proteins lack protein kinase activity and phorbol ester binding but reveal V8 peptide patterns identical to the active forms of protein kinase C (77/80 kDa) of PMA-untreated cells. The amounts of the immunoprecipitable membrane-bound 80-kDa protein kinase C-related polypeptide synthesized during the prolonged PMA treatment appear to inversely correlate with the extent of PMA-mediated growth inhibition of the respective human breast cancer cell line. These data suggest that after homologous down-regulation, functional protein kinase C (77/80 kDa) is replaced by a population of membrane-associated but enzymatically inactive protein kinase C-related polypeptides (74/80 kDa).
The spectrum of NGC 7027 (367-650 nm)
Authors: Aller Lawrence H. , Keyes Charles D.
Published: May 2006

NGC 7027 shows the richest spectrum of any known gaseous nebula. In the region of the spectrum surveyed we have measured lines of ions of H, He, C, N, O, F, Ne, Mg, Si, P, S, Cl, Ar, K, Ca, Mn, and Fe using the new Hamilton Echelle Spectrograph of Lick Observatory with a charge-coupled device (CCD) detector. Since a CCD is a linear detector, whereas photographic emulsions used in the most comprehensive previous studies were not, a considerable enhancement in the accuracy of line intensity measurements is possible.
Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII).
Authors: Pittman D D , Kaufman R J
Published: May 2006

Factor VIII functions in the intrinsic pathway of coagulation as the cofactor for factor IXa proteolytic activation of factor X. Proteolytic cleavage is required for activation and may be responsible for inactivation of cofactor activity. To identify which of the multiple cleavages are required for activation and inactivation of factor VIII, site-directed DNA-mediated mutagenesis of the factor VIII cDNA was performed and the altered forms of factor VIII were expressed in COS-1 monkey cells and characterized. Conversion of arginine residues to isoleucine residues at the aminoterminal side of the cleavage sites at positions 740, 1648, and 1721 resulted in cleavage resistance at the modified site with no alteration in the in vitro procoagulant activity and the susceptibility to thrombin activation. Similar modification of the thrombin cleavage sites at either position 372 or position 1689 resulted in molecules with residual factor VIII activity but resistant to thrombin cleavage at the modified site and not susceptible to thrombin activation. Modification of the arginine to either an isoleucine or a lysine at residue 336, the site postulated for proteolytic inactivation by activated protein C, resulted in a factor VIII molecule with increased procoagulant activity. This increased activity may result from greater resistance to proteolytic inactivation. A model for the activation and inactivation of factor VIII is proposed.
Purification and functional characterization of a cellular transcription factor that binds to an enhancer element within the adenovirus early EIIa promoter.
Authors: Jalinot P , Wintzerith M , Gaire M , Hauss C , Egly J M , Kédinger C
Published: May 2006

The adenovirus EIa-inducible early EIIa (EIIaE) promoter is comprised of several sequence elements essential for constitutive and induced expression. We report here the purification of the host-cell factor that interacts with the major upstream element of this promoter, extending between positions -90 and -70 with respect to the main EIIaE cap site and exhibiting enhancer properties. The purified factor, which corresponds to a 40- to 43-kDa polypeptide, specifically binds to its recognition site and stimulates EIIaE promoter activity when added to an in vitro transcription system, reconstituted from purified factors and RNA polymerase. The implication of this factor in the control of the other adenovirus early genes is discussed.
Human terminal deoxyribonucleotidyltransferase: molecular cloning and structural analysis of the gene and 5' flanking region.
Authors: Riley L K , Morrow J K , Danton M J , Coleman M S
Published: May 2006

Human terminal deoxyribonucleotidyltransferase (nucleoside-triphosphate:DNA deoxynucleotidylexotransferase, EC 2.7.7.31) cDNA contains an open reading frame of 1530 base pairs (bp) corresponding to a protein containing 510 amino acids. The encoded protein is a template-independent DNA polymerase found only in a restricted population of normal and malignant prelymphocytes. To begin to investigate the genetic elements responsible for the tissue-specific expression of terminal deoxyribonucleotidyltransferase, genomic clones containing the entire human gene were isolated and characterized. Initially, cDNA clones were isolated from a library generated from the human lymphoblastoid cell line, MOLT-4R. A cDNA clone containing the entire coding region of the protein was used to isolate a series of overlapping clones from two human genomic libraries. The gene comprises 11 exons and 10 introns and spans 49.4 kilobases. The 5' flanking region (709 bp) including exon 1 was sequenced. Several putative transcription initiation sites were mapped. Within 500 nucleotides of the translation start site, a series of promoter elements was detected. "TATA" and "CAAT" sequences, respectively, were found to start at nucleotides -185 and -204, -328, and 465 and -505. Start sites were found for a cyclic AMP-dependent promoter analog at nucleotide -121, an eight-base sequence corresponding to the IgG promoter enhancer (cd) at nucleotide -455, and an analog of the IgG promoter (pd) at nucleotide -159. These findings suggest that transcripts coding for terminal deoxyribonucleotidyltransferase may be variable in length and that transcription may be influenced by a variety of genetic elements.
Chloroplast DNA evolution and systematics of
Phanerophlebia
(Dryopteridaceae) and related fern genera
Authors: Yatskievych George , Stein Diana B. , Gastony Gerald J.
Published: May 2006

Restriction site variation in chloroplast DNA was examined in the neotropical fern genus
Phanerophlebia
and in selected species of the related Asiatic genus
Cyrtomium
and the cosmopolitan progenitor of these two,
Polystichum
. A total of 103 restriction site mutations was identified; these were used to construct phylogenetic networks and trees based on Wagner and Dollo parsimony and Fitch-Margoliash distance algorithms. The analyses provided evidence that
Phanerophlebia
did not arise from
Cyrtomium
. Both genera are convergent descendants from different progenitor groups in
Polystichum
, and Asiatic
Cyrtomium
is more closely related to temperate New World
Polystichum
than it is to neotropical
Phanerophlebia
. Reticulate venation, previously considered an important taxonomic character for infrageneric classification in
Phanerophlebia
, most likely evolved independently twice within the genus. Diploid maternal progenitors are suggested for two of four tetraploid species analyzed, and species-level distinctions for two closely related taxa of
Phanerophlebia
are questioned.
Intron conservation across the prokaryote-eukaryote boundary: structure of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize.
Authors: Quigley F , Martin W F , Cerff R
Published: May 2006

The nuclear gene encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from maize has been cloned and sequenced. The gene is G + C rich in its coding sequences and, in addition, contains a CpG-rich region surrounding the promoter. Further upstream several enhancer-like repetitions have been identified that may control the light- and phytochrome-mediated expression of this gene. The gene is interrupted by three introns. Introns 1 and 2 are located within the sequence encoding the transit peptide, dividing it into three parts, each containing one of the three major homology blocks typical for transit peptides of nucleus-encoded chloroplast proteins. Intron 3 is located at codon 166 (glycine) at the same nucleotide position as intron 1 in the GAPDH gene from the nematode Caenorhabditis elegans, suggesting that this intron was present in the parental GAPDH gene from which these two modern descendants originated. Intron 3 divides the GAPDH protein into its two constituent domains, the NAD-binding and the catalytic domain, immediately after helix alpha 1 at a position homologous to that of intron 9 in the gene for maize alcohol dehydrogenase, thereby confirming the prediction of Brändén et al. on the basis of gene-protein structure correlations in maize alcohol dehydrogenase for the placement of introns in the GAPDH gene [Brändén, C.I., Eklund, H., Cambillau, C. & Pryor, A.J. (1984) EMBO J. 3, 1307-1310]. These results suggest that intron 3 is an archetypical relic of early GAPDH and alcohol dehydrogenase evolution, whereas introns 1 and 2 were implicated in the evolution of chloroplast transit peptides.
Conditioning the behavior of Drosophila melanogaster by means of electric shocks.
Authors: Wallace B , Sperlich D
Published: May 2006

Eighty male Drosophila melanogaster were observed individually for extended periods (1-2 hr) as they moved about in a small chamber consisting of a narrow, clear plastic ring whose vertical walls were thin epoxy plates with electric grids by means of which an 80-V shock could be delivered to the fly. Males (whether initially subjected to 0, 1, 2, or 3 electric shocks) that were observed for 10 consecutive 5-min sessions, given a supplemental electric shock (followed by a 2-min rest), and then observed for 5 additional 5-min sessions spent only 88.9% as much time on the grids after the supplemental shock as they had been spending earlier. This avoidance of the potentially electrified grids corresponds to that observed in tests involving the exposure of flies to 0, 1, 2, or 3 electric shocks preceding the 10 5-min sessions. The results reveal (i) an extreme variation in the normal behavior among unshocked (control) males and (ii) an exponential decline in the length of time spent on the grids immediately after receiving 1, 2, or 3 shocks. These data confirm earlier reports on the conditioning of behavior in Drosophila by means of electric shocks; in addition, they remove an ambiguity inherent in the earlier studies, which relied on a presumed exposure of each of 150 or more flies to each shock as it was delivered.
Interrelations between random walks on diagrams (graphs) with and without cycles.
Authors: Hill T L
Published: May 2006

Three topics are discussed. A discrete-state, continuous-time random walk with one or more absorption states can be studied by a presumably new method: some mean properties, including the mean time to absorption, can be found from a modified diagram (graph) in which each absorption state is replaced by a one-way cycle back to the starting state. The second problem is a random walk on a diagram (graph) with cycles. The walk terminates on completion of the first cycle. This walk can be replaced by an equivalent walk on a modified diagram with absorption. This absorption diagram can in turn be replaced by another modified diagram with one-way cycles back to the starting state, just as in the first problem. The third problem, important in biophysics, relates to a long-time continuous walk on a diagram with cycles. This diagram can be transformed (in two steps) to a modified, more-detailed, diagram with one-way cycles only. Thus, the one-way cycle fluxes of the original diagram can be found from the state probabilities of the modified diagram. These probabilities can themselves be obtained by simple matrix inversion (the probabilities are determined by linear algebraic steady-state equations). Thus, a simple method is now available to find one-way cycle fluxes exactly (previously Monte Carlo simulation was required to find these fluxes, with attendant fluctuations, for diagrams of any complexity). An incidental benefit of the above procedure is that it provides a simple proof of the one-way cycle flux relation Jn +/- = IIn +/- sigma n/sigma, where n is any cycle of the original diagram.
Discrete mechanics and special relativistic random walks
Authors: Wall Frederick T.
Published: May 2006

Random walks with step lengths equal to the shortest possible physically meaningful distances are considered from the point of view of special relativity involving two observers moving uniformly with respect to each other. A requirement of statistical equivalence of the probability distributions seen by those observers leads to the Lorentz transformations, provided a randomly moving particle shifts from one submicroscopic cell of uncertainty to a neighbor with a speed equivalent to that of light. Ordinary smooth motion would appear to involve a tremendous amount of submicroscopic back and forth randomness subject to a statistical bias favoring a particular direction. The diffusive nature of the motion naturally leads to a spreading of the probability distribution.
Protein kinase activity associated with the nuclear lamina.
Authors: Dessev G , Iovcheva C , Tasheva B , Goldman R
Published: May 2006

A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea. The lamin proteins are phosphorylated on serine residues. Their two-dimensional phosphopeptide maps show multiple phosphorylation sites and a considerable similarity to the phosphopeptide maps of lamins labeled in vivo. Photoaffinity labeling experiments revealed several polypeptide fractions in the nuclear lamina fraction that are candidates for the protein kinase(s).
Direct demonstration of critical amino acid residues required for cytotoxic T-lymphocyte allorecognition of H-2 class I antigens.
Authors: McLaughlin-Taylor E , Miyada C G , McMillan M , Wallace R B
Published: May 2006

To identify critical amino acid residues recognized by alloreactive cytolytic T lymphocytes (CTL) generated between H-2Kb and H-2Kbm1, we have derived a series of cloned L-cell lines expressing the following mutant H-2Kb class I genes. Cell line L-KbTyr-Tyr expresses a mutant gene in which positions 155-156 of the Kb molecule have been changed from Arg-Leu to Tyr-Tyr, leaving position 152 unchanged. Cell line L-KbAla expresses the reciprocal mutant gene that has position 152 of the Kb molecule mutated from glutamic acid to alanine, leaving positions 155-156 unchanged. Electrophoretic mobilities of the mutant Kb molecules reflect only those changes predicted by the mutations. Mutant-specific (anti-Kbm1) and native-specific (anti-Kb) CTL lyse L-KbTyr-Tyr and L-KbAla target cells equally well. Unlabeled target inhibition of lysis revealed a pattern of recognition and inhibition that suggests that the amino acid differences between Kbm1 and Kb create at least two discrete determinants that can be recognized by different populations of CTL. The results suggest that these determinants consist, at least in part, of a linear amino acid sequence from which critical amino acid residues can be identified.
RNA polymerase II is capable of pausing and prematurely terminating transcription at a precise location in vivo and in vitro.
Authors: Resnekov O , Aloni Y
Published: May 2006

By using the minute virus of mice, we have shown that in vivo and in vitro RNA polymerase II pauses or prematurely terminates transcription at a specific location 142-147 nucleotides downstream from the P4 promoter. The attenuated RNA was found and mapped in vivo in A9 cell late after infection in both the nuclear and cytoplasmic fractions, and the terminal nucleotide was shown to have a 3' OH group. The 3' end of the attenuated RNA is capable of forming a hairpin structure that is followed by a stretch of uridines. To distinguish whether the attenuated RNA is formed as a result of processing, pausing, or termination and to dissect structural elements, factors, or mechanisms that are involved in its formation, we used in vitro systems: isolated nuclei and cell-free extracts from HeLa cells. The results of the in vitro studies show that the attenuated RNA is a result of pausing or termination and not processing. Additionally, a salt-soluble factor and RNA secondary structure were implicated in the process of termination.
Malignant transformation of human fibroblasts caused by expression of a transfected T24 HRAS oncogene.
Authors: Hurlin P J , Maher V M , McCormick J J
Published: May 2006

We showed previously that diploid human fibroblasts that express a transfected HRAS oncogene from the human bladder carcinoma cell line T24 exhibit several characteristics of transformed cells but do not acquire an infinite life-span and are not tumorigenic. To extend these studies of the T24 HRAS in human cells, we have utilized an infinite life-span, but otherwise phenotypically normal, human fibroblast cell strain, MSU-1.1, developed in this laboratory after transfection of diploid fibroblasts with a viral v-myc oncogene. Transfection of MSU-1.1 cells with the T24 HRAS flanked by two transcriptional enhancer elements (pHO6T1) yielded foci of morphologically transformed cells. No such transformation occurred if the plasmid containing T24 HRAS had only one enhancer or none at all or if the normal human HRAS gene was transfected in the pHO6 vector (pHO6N1). Cell strains derived from such foci expressed high levels of T24 HRAS product p21, formed colonies in soft agar at high frequency, proliferated rapidly in serum-free medium that does not support growth of the parental cell line, and formed progressively growing, invasive fibrosarcomas. These foci-derived T24 HRAS-transformed cell strains, as well as cells from the tumors derived from them, had the same near-diploid karyotype as that of the parental MSU-1.1 cells. Transfection of pHO6T1 into two other infinite life-span human fibroblast cell lines, cells that had not been transfected with v-myc, also resulted in malignant transformation, suggesting that the infinite life-span phenotype of MSU-1.1 cells, and not necessarily expression of the v-myc oncogene, was the factor that complemented T24 HRAS expression to cause malignant transformation.
Negative control elements and cAMP responsive sequences in the tissue-specific expression of mouse renin genes.
Authors: Nakamura N , Burt D W , Paul M , Dzau V J
Published: May 2006

The 5' flanking regions of the mouse renin genes (Ren1d and Ren2d) contain putative negative control and cAMP responsive elements. Sequence analysis shows additionally that these putative control elements in the Ren2d gene are interrupted by a 160-base-pair insertion. To document the functions of these elements, we isolated these regions and fused them to the reporter gene chloramphenicol acetyltransferase (CAT), which was linked upstream to a thymidine kinase (TK) promoter (pUTKAT1). The chimeric constructs were transfected into mouse pituitary tumor AtT-20 and human choriocarcinoma JEG-3 cells. At the basal unstimulated condition, Ren1d 5' flanking sequence in the sense orientation inhibited basal CAT expression from the TK promoter of pUTKAT1, whereas the same sequence in the antisense orientation did not. The 5' flanking region of Ren2d had no inhibitory effect on basal CAT expression. These data demonstrate that the negative control element is functional in Ren1d but is nonfunctional in Ren2d, suggesting that the 160-base-pair insertion in Ren2d interferes with the function of the negative control elements. In response to 8-bromo-cAMP, both renin genes increased transcription 3-fold, suggesting a functional cis action of the cAMP responsive element in both genes. These data may be important in the understanding of the regulation of the tissue-specific expression of mouse renin genes.
Sequence recognition protein for the 17-base-pair A + T-rich tract in the replication origin of simian virus 40 DNA.
Authors: Malkas L H , Baril E F
Published: May 2006

A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.
Vaccinia DNA topoisomerase I promotes illegitimate recombination in Escherichia coli.
Authors: Shuman S
Published: May 2006

Vaccinia virus encapsidates a Mr 32,000 type IDNA topoisomerase. Although the vaccinia gene encoding the topoisomerase is essential for virus growth, the role of the enzyme in vivo remains unclear. In the present study, the physiologic consequences of vaccinia topoisomerase action have been examined in a heterologous system, Escherichia coli. The vaccinia topoisomerase gene was inducibly expressed in an int-lambda lysogen BL21(DE3) using a T7 RNA polymerase-based transcription system. Expression of active topoisomerase in this context resulted in recA-dependent lysogenic induction as well as cell lysis. Surprisingly, topoisomerase expression also effected a 200-fold increase in the titer of infectious lambda phage, apparently by promoting int-independent prophage excision. This effect was not observed during lysogenic induction with nalidixic acid. Restriction analysis of genomic DNA from plaque-purified excisants revealed (in 10 of 10 cases) gross alterations of the DNA structure around the att site relative to the structure of the parental phage DE3. It is construed therefore that vaccinia DNA topoisomerase I acts to promote illegitimate recombination in E. coli.
A protective monoclonal antibody recognizes a linear epitope in the precursor to the major merozoite antigens of Plasmodium chabaudi adami.
Authors: Lew A M , Langford C J , Anders R F , Kemp D J , Saul A , Fardoulys C , Geysen M , Sheppard M
Published: May 2006

The monoclonal antibody 5C10/66 was shown to afford strong protection in mice against fulminating Plasmodium chabaudi adami infection. This was remarkable, as immunity to this organism is regarded to be mainly T-cell mediated. This antibody identified a 250-kDa molecule in schizonts and an 83-kDa fragment in merozoites. A cDNA clone selected by 5C10/66 was the homologue of the Plasmodium falciparum precursor to the major merozoite surface antigen (PMMSA). Comparison with the P. falciparum sequence showed that the P. chabaudi adami clone encoded the middle portion of the gene and that it can also be divided into variable and conserved blocks. Screening of a set of all possible octamer peptides predicted by the cDNA clone revealed that the core epitope of 5C10/66 was Glu-Thr-Thr-Glu-Thr. This region resides in a variable block of PMMSA.
Endothelin activates the dihydropyridine-sensitive, voltage-dependent Ca2+ channel in vascular smooth muscle.
Authors: Goto K , Kasuya Y , Matsuki N , Takuwa Y , Kurihara H , Ishikawa T , Kimura S , Yanagisawa M , Masaki T
Published: May 2006

Endothelin is a potent endothelium-derived vasoconstrictor peptide recently characterized from porcine and human vascular endothelial cells. Here we provide evidence that endothelin activates the dihydropyridine-sensitive, voltage-dependent Ca2+ channel in porcine coronary artery smooth muscle. The vasoconstrictor action of endothelin is efficiently antagonized by low doses of the dihydropyridine Ca2+-channel blocker nicardipine. Endothelin augments the Ca2+-induced contraction in a high-K+ depolarizing solution, markedly enhances high-threshold Ca2+-channel current on the whole-cell patch clamp recording, and causes a sustained increase in the intracellular Ca2+ that is largely dependent on extracellular Ca2+. These findings suggest that endothelin exerts its vasoconstrictor effect by either directly or indirectly activating the voltage-dependent Ca2+ channel.
Coevolution of host and pathogen populations in the Hordeum vulgare-Rhynchosporium secalis pathosystem.
Authors: McDonald B A , McDermott J M , Allard R W , Webster R K
Published: May 2006

Isolates of Rhynchosporium secalis collected from two experimental barley populations were scored for putative isozyme, colony color, and virulence loci. Allelic frequencies, multilocus haplotype frequencies, and multilocus genetic structure differed in the two populations of R. secalis; haplotypes also differed widely from each other in virulence. The average virulence of isolates collected from the more resistant host population was greater than the average virulence of the isolates collected from the less resistant host population; also the least virulent haplotype, which made up 19% of the pathogen population collected from the less resistant host population, accounted for only 0.3% of the isolates collected from the more resistant host population. It was concluded that the genetic systems of the barley host and fungal pathogen interacted in a complementary fashion and that the genetic structures of both the host and pathogen populations were shaped by coevolutionary processes featuring interactions among loci affecting many different traits, including interactions among host resistance genes and pathogen virulence genes.