bluuebunny/crossref_metadata_2025 huggingface dataset:
Individual chaperones required for Yop secretion by Yersinia.
Authors: Wattiau P , Bernier B , Deslée P , Michiels T , Cornelis G R
Published: May 2006

Pathogenic yersiniae secrete anti-host proteins called Yops, by a recently discovered Sec-independent pathway. The Yops do not have a classical signal peptide at their N terminus and they are not processed during membrane translocation. The secretion domain is nevertheless contained in their N-terminal part but these domains do not resemble each other in the different Yops. We have previously shown that YopE secretion requires SycE, a 15-kDa acidic protein acting as a specific cytosolic chaperone. Here we show that the gene downstream from yopH encodes a 16-kDa acidic protein that binds to hybrid proteins made of the N-terminal part of YopH and either the bacterial alkaline phosphatase or the cholera toxin B subunit. Loss of this protein by mutagenesis led to accumulation of YopH in the cytoplasm and to a severe and selective reduction of YopH secretion. This protein thus behaves like the counterpart of SycE and we called it SycH. We also engineered a mutation in lcrH, the gene upstream from yopB and yopD, known to encode a 19-kDa acidic protein. Although this mutation was nonpolar, the mutant no longer secreted YopB and YopD. The product of lcrH could be immunoprecipitated together with cytoplasmic YopD. lcrH therefore seems to encode a YopD-specific chaperone, which we called SycD. Determination of the dependence of YopB on SycD requires further investigation. SycE, SycH, and SycD appear to be members of a new family of cytosolic chaperones required for Yop secretion.
Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer.
Authors: Glover J R , Andrews D W , Rachubinski R A
Published: May 2006

The active conformation of native peroxisomal 3-ketoacyl-CoA thiolases (EC 2.3.1.16) is homodimeric. We have previously shown that a truncated Saccharomyces cerevisiae thiolase lacking its first 16 N-terminal amino acids fails to be translocated into peroxisomes but assembles into an enzymatically active form in the cytoplasm of a strain with a disrupted nuclear thiolase gene. We now report that when truncated thiolase is cosynthesized with full-length thiolase, approximately 50% of truncated thiolase cofractionates with the full-length thiolase to fractions enriched for peroxisomes and is translocated into peroxisomes as shown by its protection from the action of external proteases. We constructed an immunologically distinct cytosolic variant of thiolase by adding an influenza hemagglutinin epitope tag to the N terminus of the truncated thiolase. In a strain simultaneously expressing the full-length, truncated, and epitope-tagged truncated thiolases, we demonstrated that normally untargeted thiolase subunits are efficiently translocated into peroxisomes by dimerization with full-length thiolase subunits. Even though truncated and epitope-tagged truncated thiolase subunits are translocated into peroxisomes in this strain, only the full-length thiolase subunit can be coimmunoprecipitated with the epitope-tagged truncated thiolase subunit from the peroxisomal matrix. This observation suggests that interactions between thiolase subunits are not disrupted during translocation.
Rhythmogenesis, amplitude modulation, and multiplexing in a cortical architecture.
Authors: Kopell N , LeMasson G
Published: May 2006

In a network of excitatory and inhibitory neurons, hyperpolarization-activated inward currents can help to produce population rhythms in which individual cells participate sparsely and randomly. A shift in the activation curve of such a current changes the fraction of the cells participating in any given cycle of the population rhythm, thus changing the amplitude of the field potential. Furthermore, the frequency of the population rhythm remains relatively fixed over a substantial range of amplitudes, allowing the population rhythm to play a separate processing role from that of the individual components.
ard-1: a human gene that reverses the effects of temperature-sensitive and deletion mutations in the Escherichia coli rne gene and encodes an activity producing RNase E-like cleavages.
Authors: Wang M , Cohen S N
Published: May 2006

The Escherichia coli rne gene affects a variety of bacterial functions, including the activity of RNase E. We report the existence of a human gene (ard-1, for activator of RNA decay) that complements temperature-sensitive and deletion mutations of rne in E. coli, allowing growth of rne-defective cells, correcting abnormal cell shape, activating chemical decay of bulk mRNA, and producing site-specific cleavages characteristic of RNase E in vivo and in vitro. ard-1 encodes a highly basic 13.3-kDa proline-rich peptide that has features in common with Rne and also with eukaryotic proteins implicated in RNA binding and macromolecular transport.
Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.
Authors: Han X , Gross R W
Published: May 2006

Electrospray ionization mass spectrometry (ESI-MS) was utilized for the structural determination and quantitative analysis of individual phospholipid molecular species from subpicomole amounts of human erythrocyte plasma membrane phospholipids. The sensitivity of ESI-MS was 2-3 orders of magnitude greater than that achievable with fast-atom bombardment mass spectrometry (FAB-MS). Phospholipid structure determination and quantitative analysis with ESI-MS can be performed directly from chloroform extracts of biologic samples, obviating the need for prior chromatographic separation of phospholipid classes which has been necessary in FAB-MS phospholipid analyses. Furthermore, ESI-MS is uncomplicated by differential fragmentation of molecular ions and idiosyncratic surface desorption, allowing the quantitation of phospholipids with coefficients of determination (r2) > 0.99 and accuracies > 95%. More than 50 human erythrocyte plasma membrane phospholipid constituents were identified by direct ESI-MS analysis of chloroform extracts of plasma membranes derived from the equivalent of < 1 microliter of whole blood. The major ethanolamine glycerophospholipid subclass in erythrocyte plasma membranes was plasmenylethanolamine that was highly enriched in polyunsaturated fatty acids at the sn-2 position. Collectively, these results demonstrate that ESI-MS of phospholipids is an enabling strategy for the direct structural determination and quantitative analysis of subpicomole amounts of phospholipids from biologic samples.
Geranylgeranylated Rab proteins terminating in Cys-Ala-Cys, but not Cys-Cys, are carboxyl-methylated by bovine brain membranes in vitro.
Authors: Smeland T E , Seabra M C , Goldstein J L , Brown M S
Published: May 2006

Geranylgeranylated Rab proteins usually terminate in either Cys-Cys or Cys-Xaa-Cys, where Xaa is Ala, Ser, or Gly. In both classes of proteins, the two cysteines are geranylgeranylated, but only the Cys-Xaa-Cys class has been shown to be carboxyl-methylated on the terminal cysteine in vivo. In the current study, we used recombinant Rab geranylgeranyltransferase and a Rab escort protein (REP-1) to attach geranylgeranyl residues to the two cysteines at the carboxyl terminus of Rab3A (Cys-Ala-Cys) and Rab1A (Cys-Cys). The geranylgeranylated proteins were then incubated with bovine cerebellar membranes that contain an enzyme that transfers [3H]methyl from S-[methyl-3H]adenosyl-L-methionine to geranylgeranylated cysteine. The enzyme transferred [3H]methyl to geranylgeranylated Rab3A but not to geranylgeranylated Rab1A. Replacement of the Cys-Ala-Cys terminus of Rab3A with Cys-Cys abolished methylation, and the opposite result was obtained when the Cys-Cys of Rab1A was replaced with Cys-Ala-Cys. When the Cys-Cys terminus of Rab1A was changed to Ser-Cys, methylation was restored. These studies suggest that the carboxyl-terminal cysteine of Rab proteins terminating in Cys-Xaa-Cys but not Cys-Cys is methylated and that the resistance of Cys-Cys proteins to methylation is attributable to the vicinal geranylgeranylated cysteines.
Cloning, genomic organization, and osmotic response of the aldose reductase gene.
Authors: Ferraris J D , Williams C K , Martin B M , Burg M B , García-Pérez A
Published: May 2006

Diverse organisms accumulate organic osmolytes to adapt to hyperosmotic stress. The molecular basis of eukaryotic gene osmoregulation remains obscure. Aldose reductase [AR; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21], which catalyzes the conversion of glucose to sorbitol (an organic osmolyte), is induced in renal medullary cells under hyperosmotic conditions. Elevated extracellular NaCl increases AR mRNA transcription in PAP-HT25 cells, a cell line derived from the rabbit renal papilla. We have cloned and characterized the rabbit AR gene to determine how it is regulated by hyperosmolality. The length of the gene, not including 5' or 3' flanking regions, is approximately 14.7 kilobases (kb) organized into 10 exons and 9 introns. The transcription start site is 36 base pairs upstream of the initiator methionine codon. A 5-kb fragment containing approximately 3.5 kb of 5' flanking region was isolated. The 3.5-kb sequence was examined for basal promoter activity and hyperosmotic response in luciferase reporter gene constructs. A 235-base-pair fragment (base pairs -208 to +27) was able to drive the downstream reporter gene in transfected PAP-HT25 cells under isoosmotic conditions (300 mosmol/kg of H2O). When this fragment plus the remaining upstream sequence (from approximately base pair -3429 to base pair +27) was used, cells in hyperosmotic medium (500 mosmol/kg of H2O) showed about 40-fold induction of luciferase expression compared with cells in isoosmotic medium. The upstream fragment (from approximately base pair -3429 to base pair -192) also conferred osmotic response to a heterologous promoter (B19). This finding evidences putative osmotic response element(s) (OREs) within a specific DNA fragment in a eukaryotic genome. Identification and characterization of OREs within this fragment and their associated trans-acting factors should reveal the molecular mechanisms of gene regulation in osmotic stress.
Identification of amino acid sequence motifs in desmocollin, a desmosomal glycoprotein, that are required for plakoglobin binding and plaque formation.
Authors: Troyanovsky S M , Troyanovsky R B , Eshkind L G , Leube R E , Franke W W
Published: May 2006

By transfecting epithelial cells with gene constructs encoding chimeric proteins of the transmembrane part of the gap junction protein connexin 32 in combination with various segments of the cytoplasmic part of the desmosomal cadherin desmocollin 1a, we have determined that a relatively short sequence element is necessary for the formation of desmosome-like plaques and for the specific anchorage of bundles of intermediate-sized filaments (IFs). Deletion of as little as the carboxyl-terminal 37 aa resulted in a lack of IF anchorage and binding of the plaque protein plakoglobin, as shown by immunolocalization and immunoprecipitation experiments. In addition, we show that the sequence requirements for the recruitment of desmoplakin, another desmosomal plaque protein, differ and that a short (10 aa) segment of the desmocollin 1a tail, located close to the plasma membrane, is also required for the binding of plakoglobin, as well as of desmoplakin, and also for IF anchorage. The importance of the carboxyl-terminal domain, homologous in diverse types of cadherins, is emphasized, as it must harbor, in a mutually exclusive pattern, the information for assembly of the IF-anchoring desmosomal plaque in desmocollins and for formation of the alpha-/beta-catenin- and vinculin-containing, actin filament-anchoring plaque in E- and N-cadherin.
Characterization of ech-42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism.
Authors: Carsolio C , Gutiérrez A , Jiménez B , Van Montagu M , Herrera-Estrella A
Published: May 2006

A gene (ech-42; previously named ThEn-42) coding for one of the endochitinases produced by the biocontrol agent Trichoderma harzianum IMI206040 was cloned and characterized. Expression of the cDNA clone in Escherichia coli resulted in bacteria with chitinase activity. This chitinase has been shown to have lytic activity on Botrytis cinerea cell walls in vitro. The ech-42 gene was assigned to a double chromosomal band (chromosome V or VI) upon electrophoretic separation and Southern analysis of the chromosomes. Primer extension analysis indicated that transcription of the gene begins preferentially 109 bp upstream of the translation initiation codon. Expression of ech-42 was strongly enhanced during direct interaction of the mycoparasite with a phytopathogenic fungus when confronted in vitro and by growing it in minimal medium containing chitin as sole carbon source. Similarly, light-induced sporulation resulted in high levels of transcript, suggesting developmental regulation of the gene. The implications of these findings are discussed.
The fabJ-encoded beta-ketoacyl-[acyl carrier protein] synthase IV from Escherichia coli is sensitive to cerulenin and specific for short-chain substrates.
Authors: Siggaard-Andersen M , Wissenbach M , Chuck J A , Svendsen I , Olsen J G , von Wettstein-Knowles P
Published: May 2006

A fourth fatty acid condensing enzyme was isolated from Escherichia coli by its ability to restore elongating activity to a protein extract which had been treated with cerulenin, a condensing enzyme-specific inhibitor. The purified beta-ketoacyl-[acyl carrier protein] (ACP) synthase IV [3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41] (KAS IV) is specific for short-chain acyl-ACP substrates. The enzyme is stable at 43 degrees C and very sensitive to cerulenin (50% inhibition at 3 microM), which binds covalently. A condensing enzyme-specific antibody raised to an expressed open reading frame from barley was used to identify KAS IV protein in Western blots, and the sequence obtained for 30 amino-terminal residues. This led to the isolation of the fabJ gene located in the fab cluster at 24.8 min of the E. coli chromosome. The fabJ gene encodes a polypeptide of 413 amino acids and molecular mass 43 kDa that shows 38% identity and 64% similarity to the fabB-encoded KAS I. The amino acid sequence of KAS IV, however, is more similar to all other published condensing enzyme sequences than the KAS I sequence is. A specialized putative function for this enzyme is to supply the octanoic substrates for lipoic acid biosynthesis. We predict that an analogue of KAS IV with the same function will be found in plant mitochondria. The described complementation assay can be used to detect condensing enzymes with other substrate specificities by supplementing the cerulenin-treated extract with appropriate purified KAS enzymes.
Targeting of a distinctive protein-serine phosphatase to the protein kinase-like domain of the atrial natriuretic peptide receptor.
Authors: Chinkers M
Published: May 2006

Protein kinase-related domains of unknown function are present in the JAK family of protein tyrosine kinases and in receptor/guanylyl cyclases. I used the yeast two-hybrid system to screen for proteins interacting with the kinase-like domain of the atrial natriuretic peptide (ANP) receptor/guanylyl cyclase. A yeast strain was constructed expressing a fusion of this kinase-like domain to the lexA DNA-binding domain and containing a HIS3 gene under the control of lexA upstream activating sequences. These yeast cells were transformed with a plasmid library of mouse embryo cDNA fragments fused to the VP16 transcriptional activation domain. Cells containing VP16-fusion proteins interacting with the lexA-kinase-like domain fusion protein were selected by growth in the absence of histidine. A partial-length cDNA clone isolated by using this approach encoded a protein that interacted specifically with the ANP-receptor protein kinase-like domain both in yeast cells and in vitro. Tissue-specific expression of a 2.2-kb mRNA hybridizing to this cDNA paralleled the known pattern of ANP-receptor mRNA expression. A full-length cDNA clone isolated from a rat lung library was predicted to encode a 55-kDa protein containing at its amino terminus a targeting domain that binds to the ANP-receptor kinase-like domain and containing at its carboxyl terminus a putative protein-serine phosphatase domain. This protein is a possible candidate for the phosphatase involved in desensitizing the ANP receptor. Targeting of regulatory proteins may be an important function of protein kinase-like domains.
"Libraries from libraries": chemical transformation of combinatorial libraries to extend the range and repertoire of chemical diversity.
Authors: Ostresh J M , Husar G M , Blondelle S E , Dörner B , Weber P A , Houghten R A
Published: May 2006

The generation of diverse chemical libraries using a "libraries from libraries" concept is described. The central features of the approaches presented are the use of well-established solid-phase synthesis methods for the generation of combinatorial libraries, combined with the chemical transformation of such libraries while they remain attached to the solid support. The chemical libraries that are generated by this process have very different physical, chemical, and biological properties compared to the libraries from which they were derived. A wide range of chemical transformations are possible for peptide-based or other libraries, and an almost unlimited range of useful chemical diversities can be envisioned. In the example presented, the amide functionalities in an existing combinatorial library made up of peptides were permethylated while the library remained attached to the solid-phase support used in its synthesis. After removal of the permethylated mixtures from their solid support, this library, now lacking the typical -CONH- amide bonds of peptides, can be tested in solution with virtually all existing assay systems to identify individual compounds having specific biological activities of interest. An illustration of the use of such libraries is presented, in which the described permethylated library was used to identify individual permethylated compounds having potent antimicrobial activity against Gram-positive bacteria.
One- and two-electron oxidations of methionine by peroxynitrite.
Authors: Pryor W A , Jin X , Squadrito G L
Published: May 2006

Peroxynitrite is stable, but its acid, HOONO, either rearranges to form nitrate or oxidizes nearby biomolecules. We report here the reactions of HOONO with methionine and the methionine analog 2-keto-4-thiomethylbutanoic acid (KTBA). These oxidations proceed by two competing mechanisms. The first yields the sulfoxide; the second-order rate constants, k2, for this process for methionine and KTBA are 181 +/- 8 and 277 +/- 11 M-1.s-1, respectively, at pH 7.4 and 25 degrees C. In the second mechanism, methionine or KTBA undergoes a one-electron oxidation that ultimately gives ethylene. We propose that the one-electron oxidant is an activated form of peroxynitrous acid, HOONO*, that is formed in a steady state mechanism. The ratios of the second-order rate constants for the ethylene-producing reaction (k*2) and the first-order rate constant to produce nitric acid (kN) for methionine and KTBA, k*2/kN, are 1250 +/- 290 and 6230 +/- 1390 M-1, respectively. Both ceric and peroxydisulfate ions also oxidize KTBA to ethylene, confirming a one-electron transfer mechanism. The yields of neither MetSO nor ethylene are affected by several hydroxyl radical scavengers, suggesting that a unimolecular homolysis of HOONO to HO. and .NO2 is not involved in these reactions. HOONO* gives hydroxyl radical-like products from various substrates but displays more selectivity than does the hydroxyl radical; thus, HOONO* is incompletely trapped by typical HO. scavengers. However, a mechanism involving dissociation of HOONO* to caged radicals cannot be ruled out at this time.
Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase.
Authors: Copeland R A , Williams J M , Giannaras J , Nurnberg S , Covington M , Pinto D , Pick S , Trzaskos J M
Published: May 2006

Selective inhibition of the inducible isoform of prostaglandin G/H synthase (cyclooxygenase-2; COX2; EC 1.14.99.1) can be achieved with compounds of the general form of aryl methyl sulfonyls and aryl methyl sulfonamides. DuP 697 and NS-398 are representative examples of these compounds. Both inhibit the constitute (COX1) and inducible (COX2) isoforms of the enzyme with equal potency shortly after mixing, but their potencies increase with time for COX2 selectively. This time-dependent inhibition follows first-order kinetics, and the rate constant for inactivation of COX2 is dose dependent for both compounds. Kinetic analysis allows us to determine KI and kinact (the maximal rate of inactivation) for each inhibitor. The potency of both compounds is substrate concentration dependent, as expected for time-dependent competitive inhibitors. COX2 that has been incubated with these inhibitors, and then extensively dialyzed against buffer, shows no recovery of enzyme activity, while complete recovery of activity is seen for COX1. Thus, these inhibitors irreversibly inactivate COX2 with time, while showing minimal reversible inhibition of COX1. We isolated these inhibitors after long incubation with excess enzyme and subsequent denaturation of the enzyme. Both inhibitors showed no loss of potency resulting from interactions with COX2, suggesting that inhibition is not mediated by covalent modification of the enzyme. These data suggest that binding of these inhibitors to COX2 induces a slow structural transition of the enzyme that results in its selective inactivation.
Lipases of the euphorbiaceae family: purification of a lipase from Euphorbia characias latex and structure-function relationships with the B chain of ricin.
Authors: Moulin A , Teissère M , Bernard C , Piéroni G
Published: May 2006

A lipase from the latex of Euphorbia characias was purified using a method involving extraction with apolar solvent and adsorption chromatography on silica gel. The lipase (specific activity, 1500 international units/mg of protein) was eluted from silica gel complexes with a lipid. The main protein fraction, which had a molecular mass of 38 kDa, was inactive when dissociated from the lipid fraction. When the lipid and protein fractions were reassociated, 72% of the lipolytic activity was recovered. This lipolytic activity was inhibited by diethyl p-nitrophenyl phosphate, which was shown to bind the lipase with a molar ratio of 0.75. High specific activities (1000 international units/mg) were measured for the lipase of E. characias on lipid extracts rich in galactosyl diacylglycerols. The apolipase was sequenced up to residue 23. The B chain of ricin has a strong homology (43.5%) with that sequence and cross-reacted with antibodies raised against the purified lipase from E. characias. The activity of the B chain of ricin was comparable (54 international units/mg) to that of the apolipase of E. characias (100 international units/mg) mixed with the same lipid cofactor complex. The primary structure (residues 68-72) of the B chain of ricin contains the lipase consensus sequence Gly-Xaa-Ser-Xaa-Gly. Its reactivity with diethyl p-nitrophenyl phosphate indicates the presence of an activated serine that, in addition to its well-documented lectin activity for galactosides, suggests that the B chain of ricin may be a galactosyl diacylglycerol lipase, closely analogous to the lipase from E. characias.
Antiprogestins prevent progesterone receptor binding to hormone responsive elements in vivo.
Authors: Truss M , Bartsch J , Beato M
Published: May 2006

Antiprogestins inhibit progesterone action by competing for binding to the progesterone receptor and are potentially important pharmaceuticals in fertility control and cancer therapy. Why the complex of antiprogestins and progesterone receptor is functionally inactive is unclear. Present models are based on indirect evidence, such as transfection competition assays and in vitro DNA binding studies, partly because of difficulties in visualizing the receptor bound to DNA in vivo. Here we used genomic footprinting analysis to show ligand-dependent binding of endogenous progesterone receptor to the hormone responsive elements (HREs) of a chromosomally integrated mouse mammary tumor virus long terminal repeat in a human mammary carcinoma cell line. The antiprogestins RU 486 and ZK 98299 do not promote binding of the progesterone receptor to this natural HRE in vivo, even at concentrations that completely inhibit the agonistic effects of potent synthetic progestins. Moreover, antiprogestins cause a rapid disappearance of the agonist-induced progesterone receptor footprint. We conclude that antiprogestins interfere with receptor function by preventing its specific DNA binding.
Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice.
Authors: Champion K M , Cook R J , Tollaksen S L , Giometti C S
Published: May 2006

During the analysis of liver protein expression in the offspring of male mice irradiated with fission-spectrum neutrons, one offspring displayed a heritable 50% decrease in the abundance of two proteins. Homozygous mice lacking detectable quantities of these proteins were obtained through breeding. Characterization of this protein deficiency has identified these liver proteins as forms of the enzyme 10-formyltetrahydrofolate dehydrogenase (10-formyl-THF DH; 10-formyltetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.6). NH2-terminal sequence analysis demonstrated that both proteins share identical sequences in the first 25 residues, and this sequence matches (96% identity) that of rat and human 10-formyl-THF DH. In addition, these proteins showed cross-reactivity to polyclonal antiserum raised against purified rat 10-formyl-THF DH. Southern (DNA) blot analysis revealed a restriction fragment length polymorphism consistent with a deletion mutation in the 10-formyl-THF DH structural gene in homozygous mice. Results of Northern (RNA) blot analysis demonstrated the absence of 10-formyl-THF DH mRNA in mice lacking 10-formyl-THF DH protein. Furthermore, liver cytosolic 10-formyl-THF DH enzymatic activity was undetectable in homozygotes. Measurement of hepatic folate pools showed that in homozygotes the total folate pool is decreased and the level of tetrahydrofolate is markedly depleted.
PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major.
Authors: Bello A R , Nare B , Freedman D , Hardy L , Beverley S M
Published: May 2006

Trypanosomatid protozoans are pterin auxotrophs, a finding noted decades ago which heralded the discovery of key metabolic roles played by pteridines in eukaryotes. We have now identified the enzyme mediating unconjugated pteridine salvage in the human parasite Leishmania major, PTR1 (pteridine reductase 1, formerly hmtxr or ltdh). PTR1 is the gene in the amplified H region responsible for methotrexate (MTX) resistance, and belongs to a large family of oxidoreductases with diverse substrates and roles. We generated Leishmania lacking PTR1 by homologous gene targeting, and these ptr1- mutants required reduced biopterin (dihydro- or tetrahydrobiopterin) for growth. PTR1 purified from engineered Escherichia coli exhibited a MTX-sensitive, NADPH-dependent biopterin reductase activity. PTR1 showed good activity with folate and significant activity with dihydrofolate and dihydrobiopterin, but not with quinonoid dihydrobiopterin. PTR1 thus differs considerably from previously reported pteridine reductases of trypanosomatids and vertebrates. Pteridine reductase activity was diminished in ptr1- Leishmania and was elevated in transfected parasites bearing multiple copies of PTR1; correspondingly, ptr1- was MTX-hypersensitive whereas the multicopy transfectant was MTX-resistant. The concordance of the biochemical and genetic properties of PTR1 suggests that this is the primary enzyme mediating pteridine salvage. These findings suggest several possible mechanisms for PTR1-mediated MTX resistance and should aid in the design of rational chemotherapy.
A null mutation in the perforin gene impairs cytolytic T lymphocyte- and natural killer cell-mediated cytotoxicity.
Authors: Lowin B , Beermann F , Schmidt A , Tschopp J
Published: May 2006

Lymphocyte-mediated cytotoxicity has been proposed to consist of the polarized secretion of granule-stored perforin leading to target-cell lysis. Nevertheless, perforin-independent pathways were postulated to explain the cytolytic activity of apparently perforin-free lymphocytes and the DNA degradation found in dying target cells. To evaluate the role of perforin, we used gene targeting in embryonic stem cells to produce mice lacking perforin. Mice homozygous for the disrupted gene have no perforin mRNA. The mice are healthy. Activation and granzyme A secretion of perforin-free cytolytic T cells are unaltered. The killing activity of cytolytic T cells as well as natural killer (NK) cells, however, is impaired but not abolished. Approximately one-third of the killing activity remains when lysis of 3T3 fibroblast targets and the apoptotic cell death of YAC-1 NK targets are analyzed. We conclude that perforin is a crucial effector molecule in T cell- and NK cell-mediated cytolysis. However, alternative perforin-independent lytic mechanisms also exist.
Overexpression of human cyclin D1 reduces the transforming growth factor beta (TGF-beta) type II receptor and growth inhibition by TGF-beta 1 in an immortalized human esophageal epithelial cell line.
Authors: Okamoto A , Jiang W , Kim S J , Spillare E A , Stoner G D , Weinstein I B , Harris C C
Published: May 2006

Cyclin D1 has been implicated in G1 cell cycle progression and is frequently amplified, overtranscribed, and oversynthesized in human tumors, including esophageal carcinomas. To further address the role of cyclin D1 in cell cycle control and tumorigenesis, we have stably transfected the human cyclin D1 in the nontumorigenic esophageal epithelial cell line HET-1A. These transfected cells, which express increased amounts of cyclin D1, have enhanced colony-forming efficiency and saturation density and are resistant to growth inhibition by TGF-beta 1 compared with the parental cell line or a control vector cell clone. The clones which express increased amounts of cyclin D1 exhibited a decrease in the amount of TGF-beta type II receptor, indicating a plausible mechanism for their diminished response to TGF-beta 1. Therefore, deregulated expression of the cyclin D1 gene can modulate the negative growth factor pathway of TGF-beta 1 and may disturb the control of epithelial cell proliferation in esophageal carcinogenesis.
Three-dimensional structure of extended chromatin fibers as revealed by tapping-mode scanning force microscopy.
Authors: Leuba S H , Yang G , Robert C , Samori B , van Holde K , Zlatanova J , Bustamante C
Published: May 2006

Unfixed chicken erythrocyte chromatin fibers in very low salt have been imaged with a scanning force microscope operating in the tapping mode in air at ambient humidity. These images reveal a three-dimensional organization of the fibers. The planar "zig-zag" conformation is rare, and extended "beads-on-a-string" fibers are seen only in chromatin depleted of histones H1 and H5. Glutaraldehyde fixation reveals very similar structures. Fibers fixed in 10 mM salt appear somewhat more compacted. These results, when compared with modeling studies, suggest that chromatin fibers may exist as irregular three-dimensional arrays of nucleosomes even at low ionic strength.
The secondary structure of the ets domain of human Fli-1 resembles that of the helix-turn-helix DNA-binding motif of the Escherichia coli catabolite gene activator protein.
Authors: Liang H , Olejniczak E T , Mao X , Nettesheim D G , Yu L , Thompson C B , Fesik S W
Published: May 2006

The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known. By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site. The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet. This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor. Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins. These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins.
Evidence for a single-niche model of positive selection.
Authors: Merkenschlager M , Benoist C , Mathis D
Published: May 2006

Thymocyte maturation depends on interactions with thymic stromal elements expressing major histocompatibility complex (MHC) molecules. Mutant mouse strains lacking MHC class I (beta 2-microglobulin-null) or class II (A beta-null) expression fail to generate normal CD8 or CD4 T-cell populations and provide model systems for reconstitution experiments. We have constructed in vitro chimeras between normal and MHC-deficient thymi to evaluate the efficiency of positive selection. Unexpectedly, the generation of mature single-positive thymocytes was proportional to the fraction of wild-type (i.e., MHC-expressing) stroma over a wide range of chimerism. Similar results were obtained for the development of T-cell receptor-transgenic thymocytes in graded chimeras expressing selecting and nonselecting MHC alleles. These findings are best explained by hypothesizing that positive selection involves a rate-limiting step at which each thymocyte can interact with only one stromal cell niche.
Structure of the truncated icosahedron (such as fullerene or viral coatings) and a 60-element conjugacy class in PSl(2, 11).
Authors: Kostant B
Published: May 2006

The proper symmetry group of a truncated icosahedron P is the icosahedral group PSl(2, 5). However, knowing the symmetry group is not enough to specify the graph structure (e.g., the carbon bonds for fullerene, C60) of P. The group PSl(2, 5) is a subgroup of the 660-element group PSl(2, 11). The latter contains a 60-element conjugacy class, say M, of elements of order 11. I show here that M exhibits a model for P where the graph structure is expressed group-theoretically. For example, the 12 pentagons are the maximal commuting subsets of M. Such a model creates the opportunity of using group-based harmonic analysis (e.g., convolution calculus) to deal with problems concerning the truncated icosahedron.
Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40.
Authors: Vayssiere J L , Petit P X , Risler Y , Mignotte B
Published: May 2006

Rodent embryo cells immortalized with temperature-sensitive mutants of simian virus 40 large tumor (T) antigen have a proliferative potential that depends on temperature. At the restrictive temperature, heat-inactivation of large T antigen causes p53 release, growth arrest, and cell death. Morphological and molecular analysis indicate that the induced cell death corresponds to apoptosis. Flow cytometric analysis using a combination of forward light scatter and side scatter allows a discrimination of cells committed to apoptosis within the whole population. These cells display a reduction in cell size and a higher cellular density, confirming the apoptotic nature of the cell death. When cells exhibiting the morphological features of apoptosis were stained with a fluorescent probe of the mitochondrial membrane potential, a decreased accumulation of the dye was recorded. Measures of cellular respiration, performed with whole-cell populations, showed that the lower mitochondrial membrane potential (delta psi m) correlates, as expected, with an uncoupling of electron transport from ATP production and is linked to the induction of apoptosis. We also show that this decrease in delta psi m is associated with a decrease in the rate of mitochondrial translation. These events are detected at early stages of the apoptotic process, when most of the cells are not irreversibly committed to death, suggesting that mitochondria could be a primary target during apoptosis.
Fluctuation analysis of motor protein movement and single enzyme kinetics.
Authors: Svoboda K , Mitra P P , Block S M
Published: May 2006

We studied fluctuations in the displacement of silica beads driven by single molecules of the motor protein kinesin, moving under low mechanical loads at saturating ATP concentrations. The variance in position was significantly smaller than expected for the case of stepwise movement along a regular lattice of positions with exponentially distributed intervals. The small variance suggests that two or more sequential processes with comparable reaction rates dominate the biochemical cycle. The low value is inconsistent with certain recently proposed thermal ratchet models for motor movement as well as with scenarios where the hydrolysis of a single ATP molecule leads to a cluster of several steps. Fluctuation analysis is a potential powerful tool for studying kinetic behavior whenever the output of a single enzyme can be monitored.
Observation of binding and polymerization of Fur repressor onto operator-containing DNA with electron and atomic force microscopes.
Authors: Le Cam E , Frechon D , Barray M , Fourcade A , Delain E
Published: May 2006

The Fur (ferric uptake regulation) protein is a global regulator that, in the presence of Fe2+, represses the expression of a number of iron-acquisition genes and virulence determinants such as toxins. Dark-field electron microscopy of positively stained Fur-DNA complexes in addition to atomic force microscopy allowed direct visualization of Fur interactions with the regulatory regions of aerobactin and hemolysin operons and provided complementary information about the structure of the complexes. According to the DNA used and the protein/DNA ratio, Fur binding to DNA results in partial or total covering of the fragments, indicating that the protein initiates polymerization along the DNA molecules at specific sites. Negative staining of Fur-DNA complexes revealed a well-ordered structure of the polymer suggesting a helical arrangement. Local rigidification of the DNA molecules resulting from Fur binding could be involved in the repression process.
Light-induced isomerization causes an increase in the chromophore tilt in the M intermediate of bacteriorhodopsin: a neutron diffraction study.
Authors: Hauss T , Büldt G , Heyn M P , Dencher N A
Published: May 2006

Bacteriorhodopsin (BR) was regenerated with two selectively deuterated retinals, one with 11 deuterons in the beta-ionone ring (D11) and the other with 5 deuterons (D5) at the end of the polyene chain closest to the Schiff base at carbon atoms C-14, C-15, and C-20. Both label positions (centers of deuteration) were obtained from difference Fourier maps of projections onto the plane of the membrane by neutron diffraction at 90 K, both in the light-adapted ground-state BR568 and in the photocycle intermediate M412. To retard the decay of M412, purple membrane films were soaked in 0.1 M or 1 M guanidine hydrochloride at pH 9.6. M412 was produced by illuminating oriented membrane films at physiological temperature (278 K), followed by rapid cooling to 90 K in the absence of light. The results show that in the projected structure the ring position is unaltered during the transition from BR568 to M412, whereas the position of the D5 label shifts by 1.4 +/- 0.9 A toward the ring. The shortened interlabel distance in the projected structure for the M412 state implies that as a result of the all-trans/13-cis isomerization, the C-5 to C-13 part of the polyene chain tilts out of the plane of the membrane toward the cytoplasm by about 11 degrees +/- 6 degrees. Pairwise comparison of data sets with the same retinal for the two photocycle states M412 and BR568 leads to four difference-density maps for the protein, which are in agreement with previous work. They show changes in the protein density near helices G and F.
Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes.
Authors: Angström J , Teneberg S , Karlsson K A
Published: May 2006

Binding studies of various glycolipids, mainly belonging to the ganglio series, to the toxins isolated from Vibrio cholerae, Escherichia coli, and Clostridium tetani have been performed, using the microtiter well assay. By using the found binding preferences in conjunction with minimum-energy conformations obtained from molecular modeling of the various ligands, binding epitopes on the natural receptor glycolipids for the toxins have been defined. The binding preferences for the cholera toxin and the heat-labile E. coli toxin are very similar, with the ganglioside GM1 being the most efficient ligand. The tetanus toxin binds strongly to gangliosides of the G1b series, with GT1b as the most efficient ligand. It is found that the binding epitope on GM1 for the cholera and heat-labile toxins to a large extent overlaps with the epitope on GQ1b for the tetanus toxin.
Mutagenic potency of exocyclic DNA adducts: marked differences between Escherichia coli and simian kidney cells.
Authors: Moriya M , Zhang W , Johnson F , Grollman A P
Published: May 2006

A single-stranded shuttle vector containing a single 3,N4-etheno-2'-deoxycytidine (epsilon dC) or 1,N2-(1,3-propano)-2'- deoxyguanosine (PdG) DNA adduct was used to investigate translesional DNA synthesis in Escherichia coli and simian kidney (COS) cells. The presence of either exocyclic adduct was associated with reduced numbers of transformants. In E. coli, this inhibitory effect could be overcome partially by irradiating cells with UV light before transformation. Translesional synthesis past both exocyclic lesions was accompanied by targeted mutations. For PdG, the primary mutagenic events observed in both hosts were PdG-->T transversions; in preirradiated E. coli, PdG-->A transitions were also observed. The targeted mutation frequency for single-stranded DNA that contained PdG was 100% in nonirradiated E. coli, 68% in preirradiated cells, and 8% in COS cells. In contrast, the targeted mutation frequency for single-stranded DNA that contained epsilon dC was 2% in nonirradiated E. coli, 32% in preirradiated cells, and 81% in COS cells. The primary mutations generated by epsilon dC in both E. coli and COS cells were epsilon dC-->A and epsilon dC-->T base substitutions. These observations appear to reflect the variable specificity of DNA replication complexes in incorporating bases opposite certain adducts. We conclude that DNA synthesis past the same DNA adduct can have strikingly different consequences in bacteria and mammalian cells, underscoring the importance of establishing the intrinsic mutagenic potential of DNA adducts in mammalian cells.
Differentiation-dependent expression of the Na+/glucose cotransporter (SGLT1) in LLC-PK1 cells: role of protein kinase C activation and ongoing transcription.
Authors: Shioda T , Ohta T , Isselbacher K J , Rhoads D B
Published: May 2006

We examined changes in the mRNA level of SGLT1, a Na+/glucose cotransporter, by the differentiation status of LLC-PK1 renal epithelial cells. Proliferating (undifferentiated) cells revealed no detectable SGLT1 mRNA by Northern blot analysis. However, when cells became confluent and differentiated into polarized monolayers, there was an abrupt appearance of the SGLT1 mRNA. When confluent (differentiated) cells were dedifferentiated by reseeding at a subconfluent density, SGLT1 mRNA levels decreased quickly to nondetectable levels (t1/2 = 1.5 h), while the mRNA levels of gamma-glutamyltranspeptidase, another differentiation marker, decreased only slowly (t1/2 > 40 h). This decrease in SGLT1 mRNA was completely blocked by H-7, a protein kinase inhibitor. Since protein kinase C was highly activated in the undifferentiated cells and treatment of differentiated cells with a phorbol ester also induced quick and complete loss of SGLT1 mRNA (t1/2 = 1.5 h) but not of gamma-glutamyltranspeptidase mRNA, protein kinase C activation appears to be involved in the dedifferentiation-induced decrease in SGLT1 mRNA. Although the phorbol ester-induced decrease in the SGLT1 mRNA level was blocked completely by inhibition of transcription, inhibitors of translation blocked the decrease in mRNA levels only partially.
Initiation of recombination in Saccharomyces cerevisiae haploid meiosis.
Authors: De Massy B , Baudat F , Nicolas A
Published: May 2006

In most eukaryotes during prophase I of meiosis, homologous chromosomes pair and recombine by coordinated molecular and cellular processes. To directly test whether or not the early steps of the initiation of recombination depend on the presence of a homologous chromosome, we have examined the formation and processing of DNA double-strand breaks (DSBs, the earliest physical landmark of recombination initiation) in various haploid Saccharomyces cerevisiae strains capable of entering meiosis. We find that DSBs occur in haploid meiosis, showing that the presence of a homolog is not required for DSB formation. DSBs occur at the same positions in haploid and diploid meioses. However, these two types of meiosis exhibit subtle differences with respect to the timing of formation and levels of DSBs. In haploid meiosis, a slower rate of DSB formation and a reduction in the frequency of DSB (at one of the three sites analyzed) were observed. These results might indicate that interactions between homologs play a role in the formation of meiotic DSBs. Furthermore, haploid strains exhibit a pronounced delay in the disappearance of meiotic DSBs compared to diploid strains, which suggests that sister chromatid interactions for DSB repair are inhibited in haploid meiosis.
Interplay between excited-state intramolecular proton transfer and charge transfer in flavonols and their use as protein-binding-site fluorescence probes.
Authors: Sytnik A , Gormin D , Kasha M
Published: May 2006

A comparative study is presented of competitive fluorescences of three flavonols, 3-hydroxyflavone, 3,3',4',7-tetrahydroxyflavone (fisetin), and 4'-diethylamino-3-hydroxyflavone (DHF). The normal fluorescence S1-->S0 (400-nm region) is largely replaced by the proton-transfer tautomer fluorescence S'1-->S'0 in the 550-nm region for all three of the flavonols in aprotic solvents at room temperature. For DHF in polar solvents the normal fluorescence becomes a charge-transfer fluorescence (460-500 nm) which competes strongly with the still dominant proton-transfer fluorescence (at 570 nm). In protic solvents, and at 77 K, the interference with intramolecular hydrogen bonding gives rise to greatly enhanced normal fluorescence, lowering the quantum yield of proton-transfer fluorescence. The utility of DHF as a discriminating fluorescence probe for protein binding sites is suggested by the strong dependence of the charge-transfer fluorescence on polarity of the environment and by various static and dynamic parameters of the charge-transfer and proton-transfer fluorescence which can be determined.