bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogeny of viroids, viroidlike satellite RNAs, and the viroidlike domain of hepatitis delta virus RNA.
Authors: Elena S F , Dopazo J , Flores R , Diener T O , Moya A
Published: May 2006

We report a phylogenetic study of viroids, some plant satellite RNAs, and the viroidlike domain of human hepatitis delta virus RNA. Our results support a monophyletic origin of these RNAs and are consistent with the hypothesis that they may be "living fossils" of a precellular RNA world. Moreover, the viroidlike domain of human hepatitis delta virus RNA appears closely related to the viroidlike satellite RNAs of plants, with which it shares some structural and functional properties. On the basis of our phylogenetic analysis, we propose a taxonomic classification of these RNAs.
Heterologously expressed serotonin 1A receptors couple to muscarinic K+ channels in heart.
Authors: Karschin A , Ho B Y , Labarca C , Elroy-Stein O , Moss B , Davidson N , Lester H A
Published: May 2006

In cardiac atrial cells, muscarinic acetylcholine receptors activate a K+ current directly via a guanine nucleotide-binding protein (G protein). Serotonin type 1A receptors may activate a similar pathway in hippocampal neurons. To develop a system in which receptor/G protein/K+ channel coupling can be experimentally manipulated, we have used a highly efficient recombinant vaccinia virus vector system to express human serotonin 1A receptors in primary cultures of rat atrial myocytes. The expressed 1A receptors activated the inwardly rectifying K+ conductance that is normally activated by the endogenous muscarinic acetylcholine receptors. Maximal responses to either agonist occluded further activation by the other agonist. The average activation time constants for serotonin were about 5 times slower than for acetylcholine. The data support suggestions that the intracellular signaling pathway from seven-helix receptors to G proteins and directly to ion channels is widespread in excitable cells. After a fraction of the G proteins are activated irreversibly by guanosine 5'-[gamma-thio]triphosphate, subsequent transduction proceeds more efficiently. One possible interpretation is that multiple G-protein molecules are required to activate each channel. Vaccinia virus expression vectors are thus useful for expressing seven-helix receptors in primary cultures of postmitotic cells and have provided a heterologous expression system for the signaling pathway from seven-helix receptors to G proteins and directly to ion channels.
A phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of Caenorhabditis elegans.
Authors: Maruyama I N , Brenner S
Published: May 2006

Mutations in the unc-13 gene cause diverse defects in the nervous system of the nematode Caenorhabditis elegans. Molecular cloning of the gene and sequencing of the cDNA revealed that the product encodes a protein, 1734 amino acids in length, with a central domain with sequence similarity to the regulatory region of protein kinase C. The domain was expressed in Escherichia coli and shown to bind specifically to a phorbol ester in the presence of calcium; diacylglycerol inhibited the binding in a competitive manner. These findings confirm that the unc-13 gene product has binding sites similar to those of protein kinase C and may be a component of an alternative transduction pathway of the diacylglycerol signal to a different effector function in the nervous system.
Localization of cofactor binding sites with monoclonal anti-idiotype antibodies: phenylalanine hydroxylase.
Authors: Jennings I G , Kemp B E , Cotton R G
Published: May 2006

A monoclonal anti-idiotype antibody, NS7, previously shown to mimic the binding of the pterin cofactor of phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been used to localize the cofactor binding site within the phenylalanine hydroxylase catalytic domain to a 27-amino-acid sequence that is highly conserved among the three aromatic amino acid hydroxylases. The binding of NS7 to a synthetic peptide corresponding to the phenylalanine hydroxylase sequence from residue 263 to residue 289 was blocked by the competitive inhibitor of phenylalanine hydroxylase enzyme activity, 7,8-dihydro-6,7-dimethylpterin. In addition this peptide competed with native phenylalanine hydroxylase for binding to 6,7-dimethyl-5,6,7,8-tetrahydropterin conjugated to a polyglutamate carrier. Application of this simple and direct approach to other enzymes is likely to greatly facilitate the identification of ligand binding sites on enzymes, which will significantly contribute to the understanding of enzyme structure-function relationships.
In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: resolution of the activity into soluble and membrane-bound fractions.
Authors: Walker C J , Weinstein J D
Published: May 2006

The first committed step in chlorophyll synthesis is the Mg-chelatase-catalyzed insertion of magnesium into protoporphyrin IX. Since iron insertion into protoporphyrin leads to heme formation, Mg-chelastase lies at the branch point of heme and chlorophyll synthesis in chloroplasts. Little is known about the enzymology or regulation of Mg-chelatase, as it has been assayed only in intact cucumber chloroplasts. In this report we describe an in vitro assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts was 3- to 4-fold higher than in cucumber chloroplasts. This activity survived chloroplast lysis and could be fractionated by centrifugation into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity, and both were inactivated by boiling indicating that the enzyme is composed of soluble and membrane-bound protein(s). The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. The specific activity of the reconstituted system was typically 1 nmol of Mg-deuteroporphyrin per h per mg of protein, and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity and the enzyme was sensitive to the sulfhydryl reagent N-ethylmaleimide (I50, 20 microM). Broken and reconstituted cucumber chloroplasts were unable to maintain Mg-chelatase activity. However, the cucumber supernatant fraction was active when combined with the pellet fraction of peas; the converse was not true, which suggested that the cucumber pellet was the component that lost activity during lysis.
Triple-helix formation by alpha oligodeoxynucleotides and alpha oligodeoxynucleotide-intercalator conjugates.
Authors: Sun J S , Giovannangeli C , François J C , Kurfurst R , Montenay-Garestier T , Asseline U , Saison-Behmoaras T , Thuong N T , Hélène C
Published: May 2006

Base-pair sequences in double-stranded DNA can be recognized by homopyrimidine oligonucleotides that bind to the major groove at homopurine.homopyrimidine sequences thereby forming a local triple helix. To make oligodeoxynucleotides resistant to nucleases, we replaced the natural (beta) anomers of the nucleotide units by the synthetic (alpha) anomers. The 11-mer alpha oligodeoxynucleotide 5'-d(TCTCCTCCTTT)-3' binds to the major groove of DNA in an antiparallel orientation with respect to the homopurine strand, whereas a beta oligonucleotide adopts a parallel orientation. When an intercalating agent was attached to the 3' end of the alpha oligodeoxynucleotide, a strong stabilization of the triple helix was observed. A 16-base-pair homopurine.homopyrimidine sequence of human immunodeficiency virus proviral DNA was chosen as a target for a 16-mer homopyrimidine alpha oligodeoxynucleotide. A restriction enzyme that cleaves DNA at the junction of the homopurine.homopyrimidine sequence was inhibited by triple-helix formation. The 16-mer alpha oligodeoxynucleotide substituted by an intercalating agent was approximately 20 times more efficient than the unsubstituted oligomer. Nuclease-resistant alpha oligodeoxynucleotides offer additional possibilities to control gene expression at the DNA level.
Recombination hotspots in bacteriophage T4 are dependent on replication origins.
Authors: Yap W Y , Kreuzer K N
Published: May 2006

Bacteriophage T4 recombination "hotspots" were first detected by the rescue of genetic markers from UV-irradiated phage particles. These hotspots have since been detected following treatments that yield other forms of DNA damage, and at least one is active in the absence of damage. The previous mapping of phage replication origins near the peaks of two recombination hotspots suggested that the origins cause the localized enhancement of recombination. Here we show that deletion of one origin eliminates the corresponding recombination hotspot, as judged by rescue of markers from UV-irradiated phage. Furthermore, insertion of either origin into a recombination "coldspot" enhances rescue of nearby markers. We conclude that these origins are necessary, and very likely sufficient, for the generation of recombination hotspots. We also show that the hotspots are active in the absence of both phage-encoded UvsY and host-encoded RecA proteins, suggesting that some of the stimulated recombination occurs by a synaptase-independent mechanism.
The heme groups of cytochrome o from Escherichia coli.
Authors: Puustinen A , Wikström M
Published: May 2006

Cytochrome o, one of the two terminal ubiquinol oxidases of Escherichia coli, is structurally and functionally related to cytochrome c oxidase of mitochondria and some bacteria. It has two heme groups, one of which binds CO and forms a binuclear oxygen reaction center with copper. The other heme is unreactive toward ligands, exhibits strong interactions with the binuclear center, and is mainly responsible for the reduced-minus-oxidized alpha band. Protoheme has been thought to be the prosthetic group of b-type cytochromes, including cytochrome o. However, the hemes of cytochrome o are of a different kind, for which we propose the name heme O. Its pyridine hemochrome spectrum is blue-shifted by 4 nm relative to that of protoheme, and chromatographic behavior showed that it is much more hydrophobic than protoheme. Fast atom bombardment mass spectrometry yielded a molecular mass of 839 Da. Heme O is proposed to be a heme A-like molecule, containing a 17-carbon hydroxyethylfarnesyl side chain, but with a methyl residue replacing the formyl group.
C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.
Authors: Farnsworth C C , Kawata M , Yoshida Y , Takai Y , Gelb M H , Glomset J A
Published: May 2006

smg p25A, also known as the rab3A protein, is a small GTP-binding protein that has been implicated in intracellular vesicle transport and the secretion of neurotransmitters. It has been shown to bind reversibly to membranes, though its cDNA-predicted sequence contains no obvious membrane-binding domains. However, smg p25A does contain a cDNA-predicted C-terminal Cys-Ala-Cys sequence at positions 218 through 220, which suggests that it may be posttranslationally modified. In the present study we used two different approaches to investigate this possibility. First, we incubated pheochromocytoma cells with [3H]mevalonolactone, examined the proteins that became labeled by two-dimensional gel electrophoresis, and demonstrated that two of these proteins exactly corresponded to smg p25A. Second, we purified smg p25A from bovine brain membranes and analyzed both the full-length protein and a proteolytically derived C-terminal peptide by a combination of high performance liquid chromatography and mass spectrometry. This approach revealed that the protein's C-terminal region is methyl-esterified and contains two geranylgeranyl groups linked via thioether bonds to Cys-218 and Cys-220. Since smg p25A is one of several small GTP-binding proteins that share a C-terminal Cys-Xaa-Cys consensus sequence (where Xaa is an unspecified amino acid), our results suggest that these proteins may be similarly geranylgeranylated and methyl-esterified.
Comparative rates of desensitization of beta-adrenergic receptors by the beta-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase.
Authors: Roth N S , Campbell P T , Caron M G , Lefkowitz R J , Lohse M J
Published: May 2006

Three separate processes may contribute to rapid beta-adrenergic receptor desensitization: functional uncoupling from the stimulatory guanine nucleotide-binding protein Gs, mediated by phosphorylation of the receptors by two distinct kinases, the specific beta-adrenergic receptor kinase (beta ARK) and the cyclic AMP-dependent protein kinase A (PKA), as well as a spatial uncoupling via sequestration of the receptors away from the cell surface. To evaluate the relative importance and potential role of the various processes in different physiological situations, a kinetic analysis of these three mechanisms was performed in permeabilized A431 epidermoid carcinoma cells. To allow a separate analysis of each mechanism, inhibitors of the various desensitization mechanisms were used: heparin to inhibit beta ARK, the PKA inhibitor peptide PKI to inhibit PKA, and concanavalin A treatment to prevent sequestration. Isoproterenol-induced phosphorylation of beta 2 receptors in these cells by beta ARK occurred with a t1/2 of less than 20 sec, whereas phosphorylation by PKA had a t1/2 of about 2 min. Similarly, beta ARK-mediated desensitization of the receptors proceeded with a t1/2 of less than 15 sec, and PKA-mediated desensitization with a t1/2 of about 3.5 min. Maximal desensitization mediated by the two kinases corresponded to a reduction of the signal-transduction capacity of the receptor/adenylyl cyclase system by about 60% in the case of beta ARK and by about 40% in the case of PKA. Receptor sequestration was much slower (t1/2 of about 10 min) and involved no more than 30% of the cell surface receptors. It is concluded that beta ARK-mediated phosphorylation is the most rapid and quantitatively most important factor contributing to the rapid desensitization. This rapidity of the beta ARK-mediated mechanism makes it particularly well suited to regulate beta-adrenergic receptor function in rapidly changing environments such as the synaptic cleft.
Isoprenoid modification of rab proteins terminating in CC or CXC motifs.
Authors: Khosravi-Far R , Lutz R J , Cox A D , Conroy L , Bourne J R , Sinensky M , Balch W E , Buss J E , Der C J
Published: May 2006

Mevalonate starvation of hamster fibroblasts resulted in a shift of rab1b from the membrane to the cytosolic fraction, suggesting that rab1b depends upon an isoprenoid modification for its membrane localization. rab1b and rab3a proteins expressed in insect cells incorporated a product of [3H]mevalonate, and gas chromatography analysis of material released by Raney nickel cleavage demonstrated that rab1b and rab3a are modified by geranylgeranyl groups. Additionally, in vitro prenylation analysis demonstrated farnesyl modification of H-ras but geranylgeranyl modification of five rab proteins (1a, 1b, 2, 3a, and 6). Together, these results suggest that the carboxyl-terminal CC/CXC motifs (X = any amino acid) specifically signal for addition of geranylgeranyl, but not farnesyl, groups. A rab1b mutant protein lacking the two carboxyl-terminal cysteine residues was not prenylated in vitro. However, since a mutant H-ras protein that terminates with tandem cysteine residues was also not modified, the CC motif may be essential, but not sufficient, to signal prenylation of rab1b. Finally, rab1b and rab3a proteins were not efficient substrates for either farnesyl- or geranylgeranyltransferase activities that modify CAAX-containing proteins (A = any aliphatic amino acid). Therefore, rab proteins may be modified by a prenyltransferase(s) distinct from the prenyltransferases that modify carboxyl-terminal CAAX proteins.
Tyrosine phosphorylation provides an early and requisite signal for the activation of natural killer cell cytotoxic function.
Authors: Einspahr K J , Abraham R T , Binstadt B A , Uehara Y , Leibson P J
Published: May 2006

Natural killer (NK) cells are a unique subpopulation of lymphocytes with the capability to kill malignant cells via one of two alternative mechanisms: (i) Fc receptor-dependent cytotoxicity against antibody-coated targets or (ii) direct cell-mediated cytotoxicity. However, the molecular mechanisms that trigger and subsequently regulate NK cell cytotoxicity are incompletely understood. We have therefore investigated the role of protein tyrosine phosphorylation in the transmembrane signaling initiated after Fc receptor stimulation or direct tumor cell contact in clonal CD16+/CD3- human NK cells. We report that stimulation of the Fc receptor rapidly induced the tyrosine phosphorylation of a number of NK cell proteins. These effects occurred within 2 min, were maximal at 10 min, and declined toward baseline after 60 min. In addition, Fc receptor ligation increased the in vitro protein kinase activity of NK cell phosphotyrosyl proteins. We have also demonstrated that direct contact of NK cells with K562 tumor cells induced the rapid phosphorylation of distinct NK cell phosphotyrosyl proteins. Furthermore, the protein-tyrosine kinase inhibitor herbimycin A blocked NK cell cytotoxic function in a concentration-dependent manner. Our results suggest that protein tyrosine phosphorylation is an obligatory early proximal signal in activating the cytotoxic function of NK cells.
Restricted Epstein-Barr virus protein expression in Burkitt lymphoma is due to a different Epstein-Barr nuclear antigen 1 transcriptional initiation site.
Authors: Sample J , Brooks L , Sample C , Young L , Rowe M , Gregory C , Rickinson A , Kieff E
Published: May 2006

Epstein-Barr virus (EBV) expresses six nuclear antigens (EBNAs) and three integral latent membrane proteins (LMPs) in latently infected growth-transformed B lymphoblastoid cell lines (LCLs). In contrast, EBV protein expression in Burkitt lymphoma tissue or in newly established Burkitt lymphoma cell lines is frequently restricted to the EBV genome maintenance protein, EBNA-1. EBNA-1 expression in the absence of other EBNAs and LMP-1 has been an enigma since, in LCLs, all EBNA mRNAs are processed from a single transcript. We now show that the basis for restricted EBV expression in Burkitt lymphoma cells is selective EBNA-1 mRNA transcription from a hitherto unrecognized promoter that is 50 kb closer to the EBNA-1-encoding exon than previously described EBNA-1 promoters. Infected cells with EBNA-1-restricted expression could preferentially persist in vivo in the face of EBV-immune T-cell responses, which are frequently directed against other EBNAs and are also dependent on LMP-1 expression.
Error filtration, interference, and the human linkage map.
Authors: Shields D C , Collins A , Buetow K H , Morton N E
Published: May 2006

Typing error is a major problem in constructing human linkage maps, leading to incorrect orders and inflating map lengths. An error filter is incorporated into multiple pairwise analysis that corrects for inflation of map lengths and improves recovery of the correct order. Multipoint mapping is more sensitive to error, but when its output is adjusted for both error and interference, map lengths are no longer inflated in proportion to the number of loci and are close to those obtained by multiple pairwise analysis.
Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription.
Authors: Montgomery K F , Osborn L , Hession C , Tizard R , Goff D , Vassallo C , Tarr P I , Bomsztyk K , Lobb R , Harlan J M
Published: May 2006

Leukocyte adherence to endothelium is in part mediated by the transient expression of endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial surfaces stimulated by tumor necrosis factor alpha (TNF), interleukin (IL) 1, or bacterial lipopolysaccharide (LPS). The intracellular factors controlling induction of ELAM-1 mRNA and protein are unknown. In nuclear runoff experiments with cultured human umbilical vein endothelial cells (HUVEC), we demonstrate that transcriptional activation of the ELAM-1 gene occurs following stimulation with TNF. Sequence analysis of the 5' flanking region of the ELAM-1 gene reveals consensus DNA-binding sequences for two known transcription factors, NF-kappa B and AP-1. Gel mobility shift assays demonstrate that TNF, IL-1, or LPS (but not IL-2, IL-4, IL-6, interferon gamma, histamine, or transforming growth factor beta) induces activation of NF-kappa B-like DNA binding activity in HUVEC. In contrast, neither TNF, IL-1, nor LPS activates proteins that bind to an AP-1 consensus sequence under these experimental conditions. Phorbol 12-myristate 13-acetate, a known activator of protein kinase C (PKC), weakly induces NF-kappa B-like activity, ELAM-1 mRNA, and ELAM-1 surface expression in HUVEC. However, TNF, IL-1, and LPS do not activate PKC in HUVEC at doses that strongly induce NF-kappa B-like protein activation and ELAM-1 gene expression. PKC blockade with H7 does not inhibit activation of these NF-kappa B-like proteins but does inhibit ELAM-1 gene transcription. We conclude that PKC-independent activation of NF-kappa B in HUVEC with TNF, IL-1, or LPS is associated with, but not sufficient for, activation of ELAM-1 gene transcription.
B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2.
Authors: Gimmi C D , Freeman G J , Gribben J G , Sugita K , Freedman A S , Morimoto C , Nadler L M
Published: May 2006

Occupancy of the T-cell receptor complex does not appear to be a sufficient stimulus to induce a T-cell-mediated immune response. Increasing evidence suggests that cognate cell-cell interaction between an activated T cell and an antigen-presenting cell may provide such a stimulus. A candidate T-cell surface molecule for this costimulatory signal is the T-cell-restricted CD28 antigen. Following crosslinking with anti-CD28 mAb, suboptimally stimulated CD28+ T cells show increased proliferation and markedly increased secretion of a subset of lymphokines. Recently, the B-cell surface activation antigen B7 was shown to be a natural ligand for the CD28 molecule, and both B7 and CD28 are members of the immunoglobulin superfamily. Here we report that B7-transfected CHO cells can induce suboptimally activated CD28+ T cells to proliferate and secrete high levels of interleukin 2. The response is identical whether T cells are submitogenically stimulated with either phorbol myristate acetate or anti-CD3 to activate the T cells. This response is specific and can be totally abrogated with anti-B7 monoclonal antibody. As has previously been observed for anti-CD28 monoclonal antibody, B7 ligation induced secretion of interleukin 2 but not interleukin 4. We have previously demonstrated that B7 expression is restricted to activated B lymphocytes and interferon gamma-activated monocytes. Since these two cellular populations are involved in antigen presentation as well as cognate interaction with T lymphocytes, B7 is likely to represent a central constimulatory signal that is capable of amplifying an immune response.
A system for assaying homologous recombination at the endogenous human thymidine kinase gene.
Authors: Benjamin M B , Potter H , Yandell D W , Little J B
Published: May 2006

A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK+/+ parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or -1 frameshifts. Resulting TK-/- mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. Mutations were characterized by exon-specific polymerase chain reaction amplification and direct sequencing. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by approximately 8 kilobases. These lines undergo spontaneous reversion to TK+ at a frequency of less than 10(-7), and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK+. The mode of reversion to TK+ was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. Our data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.
Correlation between retinoblastoma gene expression and differentiation in human testicular tumors.
Authors: Strohmeyer T , Reissmann P , Cordon-Cardo C , Hartmann M , Ackermann R , Slamon D
Published: May 2006

Inactivation of the retinoblastoma gene (RB gene) is associated with the development of several human malignancies including retinoblastomas, some osteo- and soft tissue sarcomas, small cell lung cancer, and possibly breast and bladder cancers. To our knowledge, this gene has not been evaluated in human germ-cell malignancies. In this study 67 primary testicular germ-cell tumors and 4 testicular non-germ-cell malignancies were examined to determine the prevalence and nature of RB gene alterations. Decreased expression of RB gene mRNA was found in all testicular germ-cell tumors (both seminomas and nonseminomas) examined. The RB protein could not be detected by immunohistochemical analysis in the undifferentiated cells of any germ-cell tumors whereas the differentiated malignant cells present in 14/15 teratocarcinomas expressed the protein. No gross alterations of the RB gene were found at DNA level in any of the examined specimens. This and the presence of the RB protein in the more differentiated tumor cells of teratocarcinomas suggest that changes in transcript levels rather than mutation(s) of the gene may be responsible for the absent or decreased RB expression in human germ-cell tumors. To date studies on the mechanism of RB regulation have demonstrated that it occurs at the protein level by phosphorylation of the p105 gene product. The findings presented here indicate that additional regulation might occur at the transcript level.
Induction of soybean vegetative storage proteins and anthocyanins by low-level atmospheric methyl jasmonate.
Authors: Franceschi V R , Grimes H D
Published: May 2006

Soybean seedlings were exposed to atmospheric methyl jasmonate (MJ) to determine if low levels of this compound could regulate the expression and accumulation of the vegetative storage proteins (VSPs) in soybeans. Low levels of atmospheric MJ induced the accumulation of three VSPs with molecular masses of 27 kDa, 29 kDa, and 94 kDa (vsp27, vsp29, and vsp94, respectively). Atmospheric MJ caused vsp94 to be accumulated in all above-ground organs of the seedling uniformly after just 3 days of exposure. vsp27 preferentially accumulated in shoot tips and primary leaves, whereas vsp29 preferentially accumulated in the cotyledons. In addition to these effects, MJ also induced the biosynthesis of anthocyanins in light-grown seedlings but inhibited anthocyanin biosynthesis in etiolated seedlings. It is concluded that low levels of atmospheric MJ regulate anthocyanin biosynthesis and the organspecific accumulation of VSPs in developing soybean seedlings. The organ-specific differential accumulation may reflect changes in the pattern of nitrogen partitioning between various compounds and/or organs. These results lend substance to the hypothesis that volatile MJ may act as a gaseous messenger or growth regulator in plants.
Drosophila Rrp1 protein: an apurinic endonuclease with homologous recombination activities.
Authors: Sander M , Lowenhaupt K , Rich A
Published: May 2006

A protein previously purified from Drosophila embryo extracts by a DNA strand transfer assay, Rrp1 (recombination repair protein 1), has an N-terminal 427-amino acid region unrelated to known proteins, and a 252-amino acid C-terminal region with sequence homology to two DNA repair nucleases, Escherichia coli exonuclease III and Streptococcus pneumoniae exonuclease A, which are known to be active as apurinic endonucleases and as double-stranded DNA 3' exonucleases. We demonstrate here that purified Rrp1 has apurinic endonuclease and double-stranded DNA 3' exonuclease, activities and carries out single-stranded DNA renaturation in a Mg(2+)-dependent manner. Strand transfer, 3' exonuclease, and single-stranded DNA renaturation activities comigrate during column chromatography. The properties of Rrp1 suggest that it could promote homologous recombination at sites of DNA damage.
Prediction of optimal peptide mixtures to induce broadly neutralizing antibodies to human immunodeficiency virus type 1.
Authors: Holley L H , Goudsmit J , Karplus M
Published: May 2006

Sequences of the principal neutralizing determinant (PND) of the external envelope protein, gp120, from 245 isolates of human immunodeficiency virus type 1 are analyzed. The minimal set of peptides that would elicit antibodies to neutralize a majority of U.S. and European isolates of human immunodeficiency virus type 1 is determined with the assumption that peptides of a given length including the central Gly-Pro-Gly triad are required. In spite of the hypervariability of the PND, 90% of these 245 sequences include peptides from a set of 7 pentapeptides, 13 hexapeptides, or 17 heptapeptides. Tests of these peptide sets on 78 additional PND sequences show that 95% are covered by the 7 pentapeptides, 94% by the 13 hexapeptides, and 86% by the 17 heptapeptides. To anticipate variants not yet observed, single amino acid mutation frequencies from the 245 isolates are used to calculate an expanded set of the 10,000 most probable PND sequences. These sequences cover 86% of the total distribution expected for the central portion of the PND. Peptide lists derived from this expanded set when tested on the 78 additional sequences show that 7 pentapeptides cover 95%, 13 hexapeptides cover 94%, and 17 heptapeptides cover 94%. These results suggest that peptide cocktails of limited size with the potential to cover a large fraction of PND sequence variation may be feasible vaccine candidates.
Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase.
Authors: Banci L , Bertini I , Turano P , Tien M , Kirk T K
Published: May 2006

Lignin peroxidase shares several structural features with the well-studied horseradish peroxidase and cytochrome c peroxidase but carries a higher redox potential. Here the heme domain of lignin peroxidase and the lignin peroxidase cyanide adduct was examined by 1HNMR spectroscopy, including nuclear Overhauser effect and two-dimensional measurements, and the findings were compared with those for horseradish peroxidase and cytochrome c peroxidase. Structural information was obtained on the orientation of the heme vinyl and propionate groups and the proximal and distal histidines. The shifts of the epsilon1 proton of the proximal histidine were found to be empirically related to the Fe3+/Fe2+ redox potentials.
Static and statistical bending of DNA evaluated by Monte Carlo simulations.
Authors: Zhurkin V B , Ulyanov N B , Gorin A A , Jernigan R L
Published: May 2006

To investigate the influence of thermal fluctuations on DNA curvature the Metropolis procedure at 300 K was applied to B-DNA decamers containing A5.T5 and A4.T4 blocks. Monte Carlo simulations have confirmed the DNA bending anisotropy: B-DNA bends most easily in a groove direction (roll). The A5.T5 block is more rigid than the other sequences; the pyrimidine-purine dimers are found to be the most flexible. For A5TCTCT, A5CTCTC, and A5GAGAG, the average bend angle per decamer is 20-25 degrees in a direction toward the minor groove in the center of the A5.T5 tract, which is consistent with both the "junction" and "wedge AA" models. However, in A5T5, A4T4CG, and T4A4GC, bending is directed into the grooves at the 5' and 3' ends of purine tracts. Thus, directionality of bending caused by An.Tn blocks strongly depends on their neighboring sequences. These calculations demonstrate that the sequence-dependent variation of the minor-groove width mimics the observed hydroxyl radical cleavage pattern. To estimate the effect of fluctuations on the overall shape of curved DNA fragments, longer pieces of DNA (up to 200 base pairs) were generated. For sequences with strong curvature (A5X5 and A4T4CG), the static model and Monte Carlo ensemble give similar results but, for moderately and slightly curved sequences (A5T5 or T4A4GC), the static model predicts a much smaller degree of bending than does the statistical representation. Considering fluctuations is important for quantitative interpretation of the gel electrophoresis measurements of DNA curvature, where both the static and statistical bends are operative.
Single-unit and 2-deoxyglucose studies of side inhibition in macaque striate cortex.
Authors: Born R T , Tootell R B
Published: May 2006

In the course of studies to map spatial frequency tuning of neurons in layers 2 and 3 of macaque striate cortex, we found that a high proportion (70%) of cells in the interblob regions responded poorly to full-field gratings, compared with responses to single bars, edges, or delimited gratings. This was most often due to side inhibition, in which increasing the number of cycles of a grating placed within the cell's receptive field causes progressive inhibition of response. Quantitative receptive-field mappings showed, however, that the inhibition can occur within the region activated by a bar, as well as beyond it. The inhibition appears to be orientation-selective, in that a surround grating was more effective at inhibiting the response to a center grating patch if it was of similar orientation. 2-Deoxyglucose experiments confirmed that side inhibition is very widespread in the interblobs of layers 2 and 3 and suggested that it is reduced or lacking in layers 4A through 6. Since layers 2 and 3 of striate cortex are the major source of cortical projections to area V2 and beyond, the prevalence of side stopping in these laminae has implications for theories of cortical visual function. Side-stopped interblob cells may be acting as "contour-pass filters" that filter out redundant information in textured or noisy surfaces, focusing subsequent form processing on contrasts corresponding to object boundaries.
Tetrahydrobiopterin, a cofactor for rat cerebellar nitric oxide synthase, does not function as a reactant in the oxygenation of arginine.
Authors: Giovanelli J , Campos K L , Kaufman S
Published: May 2006

Studies with purified nitric oxide synthase from rat cerebellum have confirmed previous reports that product formation is enhanced by tetrahydrobiopterin [H4B; 6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin]. The effect of the natural isomer, (6R)-H4B, is observed at extremely low (less than 0.1 microM) concentrations and is remarkably selective. At these concentrations, only the diastereoisomer (6S)-H4B, the structural isomer 7-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin, and 7,8-dihydrobiopterin showed detectable effects. Our observations are inconsistent with a stoichiometric role for H4B in the oxygenation of arginine [e.g., Stuehr, D. J., Kwon, N. S., Nathan, C. F., Griffith, O. W., Feldman, P. L. & Wiseman, J. (1991) J. Biol. Chem. 266, 6259-6263]. Activity is initially independent of added H4B; enhanced product formation with H4B is observed only as incubation progresses. The effect of H4B is catalytic, with each mole of added H4B supporting the formation of greater than 15 mol of product. Recycling of H4B was excluded by direct measurement during nitric oxide synthesis and by the demonstration that nitric oxide synthase is not inhibited by methotrexate. These combined results exclude H4B as a stoichiometric reactant and suggest that H4B enhances product formation by protecting enzyme activity against progressive loss. Preliminary studies indicate that the decreased activity in the absence of added H4B does not depend on catalytic turnover of the enzyme. The role of H4B may be allosteric or it may function to maintain some group(s) on the enzyme in a reduced state required for activity.
Transforming growth factor beta stimulates urokinase-type plasminogen activator and DNA synthesis, but not prostaglandin E2 production, in human synovial fibroblasts.
Authors: Hamilton J A , Piccoli D S , Leizer T , Butler D M , Croatto M , Royston A K
Published: May 2006

Transforming growth factor beta (TGF-beta) is usually associated with matrix formation and tissue repair; in contrast, cellular expression of the serine proteinase, urokinase-type plasminogen activator (u-PA) is often correlated with tissue remodeling, as well as with cell migration and transformation. We report here that purified recombinant human TGF-beta (greater than or equal to 300 pg/ml) can stimulate rapidly (within 2 h) the u-PA activity of nonrheumatoid synovial fibroblast-like cells. As for interleukin 1 (IL-1), u-PA mRNA levels are raised in response to TGF-beta, but unlike IL-1, no increase in prostaglandin E2 levels occurs. In contrast to a number of other examples in the literature, in which these two cytokines have opposing actions, TGF-beta can potentiate the action of optimal concentrations of IL-1 in enhancing u-PA expression. These effects of TGF-beta are similar to those of all-trans-retinoic acid. In addition, synovial fibroblast DNA synthesis was stimulated by TGF-beta. Because TGF-beta has been detected in the synovia of patients with rheumatoid arthritis and has been shown to reduce the collagenase levels and proliferation of synovial fibroblast-like cells, it has been proposed by others to be involved beneficially in the reparative processes occurring in arthritic lesions. However, on the basis of our findings, we propose alternative functions for this cytokine--namely, roles in the destructive events as well as in the synovial hyperplasia observed in rheumatoid joints.
cis-acting DNA elements responsive to gibberellin and its antagonist abscisic acid.
Authors: Skriver K , Olsen F L , Rogers J C , Mundy J
Published: May 2006

We have used a transient expression assay in aleurone protoplasts of barley to delineate hormone response elements of the abscisic acid (ABA)-responsive rice gene Rab16A and of the gibberellin A3 (GA3)-responsive barley alpha-amylase gene Amy 1/6-4. Our approach used transcriptional fusions between their 5' upstream sequences and a bacterial chloramphenicol acetyltransferase reporter gene. A chimeric promoter containing six copies of the -181 to -171 region of Rab 16A fused to a minimal promoter conferred ABA-responsive expression on the reporter gene. Transcription from this ABA response element (GTACGTGGCGC) was unaffected by GA3. A chimeric promoter containing six copies of the -148 to -128 sequence of Amy 1/6-4 fused to the minimal promoter conferred GA3-responsive expression on the reporter gene. Transcription from this GA3 response element (GGCCGATAACAAACTCCGGCC) was repressed by ABA. The effect on transcription from both hormone response elements was orientation-independent, indicating that they function as inducible enhancers in their native genes.
A single amino acid substitution strongly modulates the activity and substrate specificity of the mouse mdr1 and mdr3 drug efflux pumps.
Authors: Gros P , Dhir R , Croop J , Talbot F
Published: May 2006

Specific protein domains and amino acids responsible for the apparent capacity of P-glycoprotein (mdr) to recognize and transport a large group of structurally unrelated drugs have not been identified. We have introduced a single Ser----Phe substitution within the predicted TM11 domain of mdr1 (position 941) and mdr3 (position 939) and analyzed the effect of these substitutions on the drug-resistance profiles of these two proteins. Mutations at this residue drastically altered the overall degree of drug resistance conveyed by mdr1 and mdr3. The modulating effect of this mutation on mdr1 and mdr3 varied for the drugs tested: it was very strong for colchicine and adriamycin and moderate for vinblastine. For mdr1, the Ser941----Phe941 substitution produced a unique mutant protein that retained the capacity to confer vinblastine resistance but lost the ability to confer adriamycin and colchicine resistance. These results strongly suggest that the predicted TM11 domain of proteins encoded by mdr and mdr-like genes plays an important role in the recognition and transport of their specific substrates.
The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme.
Authors: Dohmen R J , Madura K , Bartel B , Varshavsky A
Published: May 2006

The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Distinct versions of the N-end rule operate in all organisms examined, from mammals to bacteria. We show that UBC2(RAD6), one of at least seven ubiquitin-conjugating enzymes in the yeast Saccharomyces cerevisiae, is essential for multiubiquitination and degradation of the N-end rule substrates. We also show that UBC2 is physically associated with UBR1, the recognition component of the N-end rule pathway. These results indicate that some of the UBC2 functions, which include DNA repair, induced mutagenesis, sporulation, and regulation of retrotransposition, are mediated by protein degradation via the N-end rule pathway.
Cytoduction in Chlamydomonas reinhardtii.
Authors: Matagne R F , Remacle C , Dinant M
Published: May 2006

After conjugation between Chlamydomonas gametes of opposite mating type, a transient dikaryon is formed. The two nuclei fuse within 4-6 hr after mating. The young diploid zygote differentiates into dormant zygospore competent to complete meiosis, or more rarely (2-10% of cases) it undergoes mitosis to produce a stable diploid progeny. We here bring genetical, biochemical, and cytological evidence that among the mitotic zygotes, a large proportion of them undergo cytokinesis without fusion of the nuclei-a process that has been termed "cytoduction." By using appropriate genetic markers, haploid cytoductants that possess the nuclear genotype of one parent and the chloroplast marker of the other parent can easily be isolated. Genetical analysis and hybridization experiments moreover show that many haploid cytoductants transmit the chloroplast DNA molecules of both parents and that, as in diploids, these DNA copies occasionally recombine. This process of cytoduction extends the life cycle of Chlamydomonas and provides new tools for its genetic analysis.
Functional significance of an overlapping consensus binding motif for Sp1 and Zif268 in the murine adenosine deaminase gene promoter.
Authors: Ackerman S L , Minden A G , Williams G T , Bobonis C , Yeung C Y
Published: May 2006

The murine adenosine deaminase (ADA) gene has a (G + C)-rich promoter that can support diverse tissue-specific gene expression. By using deletion and mutation analyses, we have identified a cis-acting "repressor" element located immediately upstream of the proximal Sp1 binding site in the ADA gene promoter. This repressor element was localized to a binding site for the immediate-early, serum-responsive, DNA binding factor Zif268. This Zif268 binding site partially overlaps a binding site for the general transcription activator Sp1. Disruption of the Zif268 binding site without disturbing the Sp1 binding motif abolished Zif268 binding and resulted in significantly elevated promoter function. Conversely, disruption of the proximal consensus Sp1 binding motif without disturbing the Zif268 binding site resulted in a loss of Sp1 binding at that region and greatly reduced promoter activity. Our results suggest that one function of Zif268 may be to down-regulate this type of mammalian gene promoter by competing with Sp1 for binding to the overlapping binding motif.
Detection of human T-lymphotropic virus-like particles in cultures of peripheral blood lymphocytes from patients with mycosis fungoides.
Authors: Zucker-Franklin D , Coutavas E E , Rush M G , Zouzias D C
Published: May 2006

Because of seronegativity and absence of a leukemic phase in most patients with mycosis fungoides, a role for the human T-lymphotropic virus type I (HTLV-I) in this disease has remained tenuous. Virus particles are not seen in fresh isolates of skin or blood lymphocytes and the malignant cells (Sézary cells) have been difficult to culture. The availability of growth factors and biomolecular techniques have prompted a renewed attempt to find evidence of virus infection in these patients. We report here the successful culture of blood lymphocytes of 17 patients with mycosis fungoides and 1 patient with the Sézary syndrome. The cells of 2 additional patients failed to grow after 4-6 weeks in vitro. Ultrastructural analysis of the cultures showed an abundance of HTLV-like particles in the specimens of 18 of the 20 patients. Preliminary immunohistochemical studies carried out with various antisera directed against HTLV-I and the polymerase chain reaction utilizing a probe for a conserved region of the pol gene of HTLV-I were positive on only a portion of the specimens. Although definitive characterization of this organism awaits further analysis, it seems likely that circulating lymphocytes of all patients with mycosis fungoides harbor a virus that morphologically resembles HTLV-I.
Chicken T-cell receptor beta-chain diversity: an evolutionarily conserved D beta-encoded glycine turn within the hypervariable CDR3 domain.
Authors: McCormack W T , Tjoelker L W , Stella G , Postema C E , Thompson C B
Published: May 2006

Unlike mammals, chickens generate an immunoglobulin (Ig) repertoire by a developmentally regulated process of intrachromosomal gene conversion, which results in nucleotide substitutions throughout the variable regions of the Ig heavy- and light-chain genes. In contrast to chicken Ig genes, we show in this report that diversity of the rearranged chicken T-cell receptor (TCR) beta-chain gene is generated by junctional heterogeneity, as observed in rearranged mammalian TCR genes. This junctional diversity increases during chicken development as a result of an increasing base-pair addition at the V beta-D beta and D beta-J beta joints (where V, D, and J are the variable, diversity, and joining gene segments). Despite the junctional hypervariability, however, almost all functional V beta-D beta-J beta junctions appear to encode a glycine-containing beta-turn. Such a turn may serve to position the amino acid side chains of a hypervariable TCR beta-chain loop with respect to the antigen-binding groove of the major histocompatibility complex molecule. Consistent with this hypothesis, the germ-line D beta nucleotide sequences of chickens, mice, rabbits, and humans have been highly conserved and encode a glycine in all three reading frames.