Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription

Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed.

Archaeological evidence of teosinte domestication from Guilá Naquitz, Oaxaca

Analysis of the three most ancient Zea mays inflorescence fragments from Guilá Naquitz, Oaxaca, Mexico shows they did not disarticulate naturally, indicating that agricultural selection of domesticated teosinte was underway by 5,400 14C years before the present (about 4,200 dendrocalibrated years B.C.). The cooccurrence of two-ranked specimens with two rows and four rows of grain and numerous additional morphological characteristics of these specimens support hypotheses based on molecular and quantitative genetic analyses that maize evolved from teosinte. Domestication of the wild ancestor of maize occurred before the end of the 5th millennium B.C.

Structure of the thrombin complex with triabin, a lipocalin-like exosite-binding inhibitor derived from a triatomine bug

Triabin, a 142-residue protein from the saliva of the blood-sucking triatomine bug Triatoma pallidipennis, is a potent and selective thrombin inhibitor. Its stoichiometric complex with bovine α-thrombin was crystallized, and its crystal structure was solved by Patterson search methods and refined at 2.6-Å resolution to an R value of 0.184. The analysis revealed that triabin is a compact one-domain molecule essentially consisting of an eight-stranded β-barrel. The eight strands A to H are arranged in the order A-C-B-D-E-F-G-H, with the first four strands exhibiting a hitherto unobserved up-up-down-down topology. Except for the B-C inversion, the triabin fold exhibits the regular up-and-down topology of lipocalins. In contrast to the typical ligand-binding lipocalins, however, the triabin barrel encloses a hydrophobic core intersected by a unique salt-bridge cluster. Triabin interacts with thrombin exclusively via its fibrinogen-recognition exosite. Surprisingly, most of the interface interactions are hydrophobic. A prominent exception represents thrombin’s Arg-77A side chain, which extends into a hydrophobic triabin pocket forming partially buried salt bridges with Glu-128 and Asp-135 of the inhibitor. The fully accessible active site of thrombin in this complex is in agreement with its retained hydrolytic activity toward small chromogenic substrates. Impairment of thrombin’s fibrinogen converting activity or of its thrombomodulin-mediated protein C activation capacity upon triabin binding is explained by usage of overlapping interaction sites of fibrinogen, thrombomodulin, and triabin on thrombin. These data demonstrate that triabin inhibits thrombin via a novel and unique mechanism that might be of interest in the context of potential therapeutic applications.

Thioredoxin-linked mitigation of allergic responses to wheat

Thioredoxin, a ubiquitous 12-kDa regulatory disulfide protein, was found to reduce disulfide bonds of allergens (convert S—S to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the protein fractions extracted from wheat flour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the α and β fractions were most potent, followed by the γ and ω types. Thioredoxin mitigated the allergenicity associated with the major protein fractions—i.e, the gliadins (including the α, β, and γ types) and the glutenins—but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP–thioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) disulfide bonds. The present results demonstrate that the reduction of these disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active proteins. This decrease occurs alongside the changes identified previously—i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, more-digestible foods.

Conformational changes in tertiary structure near the ligand binding site of an integrin I domain

For efficient ligand binding, integrins must be activated. Specifically, a conformational change has been proposed in a ligand binding domain present within some integrins, the inserted (I) domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333–1340]. This proposal remains controversial, however, despite extensive crystal structure studies on the I domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333–1340; Liddington, R. & Bankston, L. (1998) Structure (London) 6, 937–938; Qu, A. & Leahy, D. J. (1996) Structure (London) 4, 931–942; and Baldwin, E. T., Sarver, R. W., Bryant, G. L., Jr., Curry, K. A., Fairbanks, M. B., Finzel, B. C., Garlick, R. L., Heinrikson, R. L., Horton, N. C. & Kelly, L. L. (1998) Structure (London) 6, 923–935]. By defining the residues present in the epitope of a mAb against the human Mac-1 integrin (αMβ2, CD11b/CD18) that binds only the active receptor, we provide biochemical evidence that the I domain itself undergoes a conformational change with activation. This mAb, CBRM1/5, binds the I domain very close to the ligand binding site in a region that is widely exposed regardless of activation as judged by reactivity with other antibodies. The conformation of the epitope differs in two crystal forms of the I domain, previously suggested to represent active and inactive receptor. Our data suggests that conformational differences in the I domain are physiologically relevant and not merely a consequence of different crystal lattice interactions. We also demonstrate that the transition between the two conformational states depends on species-specific residues at the bottom of the I domain, which are proposed to be in an interface with another integrin domain, and that this transition correlates with functional activity.

What was natural in the coastal oceans?

Humans transformed Western Atlantic coastal marine ecosystems before modern ecological investigations began. Paleoecological, archeological, and historical reconstructions demonstrate incredible losses of large vertebrates and oysters from the entire Atlantic coast. Untold millions of large fishes, sharks, sea turtles, and manatees were removed from the Caribbean in the 17th to 19th centuries. Recent collapses of reef corals and seagrasses are due ultimately to losses of these large consumers as much as to more recent changes in climate, eutrophication, or outbreaks of disease. Overfishing in the 19th century reduced vast beds of oysters in Chesapeake Bay and other estuaries to a few percent of pristine abundances and promoted eutrophication. Mechanized harvesting of bottom fishes like cod set off a series of trophic cascades that eliminated kelp forests and then brought them back again as fishers fished their way down food webs to small invertebrates. Lastly, but most pervasively, mechanized harvesting of the entire continental shelf decimated large, long-lived fishes and destroyed three-dimensional habitats built up by sessile corals, bryozoans, and sponges. The universal pattern of losses demonstrates that no coastal ecosystem is pristine and few wild fisheries are sustainable along the entire Western Atlantic coast. Reconstructions of ecosystems lost only a century or two ago demonstrate attainable goals of establishing large and effective marine reserves if society is willing to pay the costs. Historical reconstructions provide a new scientific framework for manipulative experiments at the ecosystem scale to explore the feasibility and benefits of protection of our living coastal resources.

Targeted mutation of the murine arylhydrocarbon receptor nuclear translocator 2 (Arnt2) gene reveals partial redundancy with Arnt

The ubiquitously expressed basic helix–loop–helix (bHLH)-PAS protein ARNT (arylhydrocarbon receptor nuclear transporter) forms transcriptionally active heterodimers with a variety of other bHLH-PAS proteins, including HIF-1α (hypoxia-inducible factor-1α) and AHR (arylhydrocarbon receptor). These complexes regulate gene expression in response to hypoxia and xenobiotics, respectively, and mutation of the murine Arnt locus results in embryonic death by day 10.5 associated with placental, vascular, and hematopoietic defects. The closely related protein ARNT2 is highly expressed in the central nervous system and kidney and also forms complexes with HIF-1α and AHR. To assess unique roles for ARNT2 in development, and reveal potential functional overlap with ARNT, we generated a targeted null mutation of the murine Arnt2 locus. Arnt2−/− embryos die perinatally and exhibit impaired hypothalamic development, phenotypes previously observed for a targeted mutation in the murine bHLH-PAS gene Sim1 (Single-minded 1), and consistent with the recent proposal that ARNT2 and SIM1 form an essential heterodimer in vivo [Michaud, J. L., DeRossi, C., May, N. R., Holdener, B. C. & Fan, C. (2000) Mech. Dev. 90, 253–261]. In addition, cultured Arnt2−/− neurons display decreased hypoxic induction of HIF-1 target genes, demonstrating formally that ARNT2/HIF-1α complexes regulate oxygen-responsive genes. Finally, a strong genetic interaction between Arnt and Arnt2 mutations was observed, indicating that either gene can fulfill essential functions in a dose-dependent manner before embryonic day 8.5. These results demonstrate that Arnt and Arnt2 have both unique and overlapping essential functions in embryonic development.

Specific Soybean Lipoxygenases Localize to Discrete Subcellular Compartments and Their mRNAs Are Differentially Regulated by Source-Sink Status1

Members of the lipoxygenase multigene family, found widely in eukaryotes, have been proposed to function in nitrogen partitioning and storage in plants. Lipoxygenase gene responses to source-sink manipulations in mature soybean (Glycine max [L.] Merr.) leaves were examined using gene-specific riboprobes to the five vegetative lipoxygenases (vlxA–vlxE). Steady-state levels of all vlx mRNAs responded strongly to sink limitation, but specific transcripts exhibited differential patterns of response as well. During reproductive sink limitation, vlxA and vlxB messages accumulated to high levels, whereas vlxC and vlxD transcript levels were modest. Immunolocalization using peptide-specific antibodies demonstrated that under control conditions, VLXB was present in the cytosol of the paraveinal mesophyll and with pod removal accumulated additionally in the bundle-sheath and adjacent cells. With sink limitation VLXD accumulated to apparent high levels in the vacuoles of the same cells. Segregation of gene products at the cellular and subcellular levels may thus permit complex patterns of differential regulation within the same cell type. Specific lipoxygenase isoforms may have a role in short-term nitrogen storage (VLXC/D), whereas others may simultaneously function in assimilate partitioning as active enzymes (VLXA/B).

In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains.

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death. Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in ail known ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (Kj = 14 nM) by the tetrapeptide aldehyde DEVD-CHO. Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathways.

Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene.

Sterol-regulated transcription of the gene for rat farnesyl diphosphate (FPP) synthase (geranyl-diphosphate:isopentenyl-diphosphate geranyltranstransferase, EC 2.5.1.10) is dependent in part on the binding of the ubiquitous transcription factor NF-Y to a 6-bp element within the proximal promoter. Current studies identify a second element in this promoter that is also required for sterol-regulated transcription in vivo. Mutation of three nucleotides (CAC) within this element blocks the 8-fold induction of FPP synthase promoter-reporter genes that normally occurs when the transfected cells are incubated in medium deprived of sterols. Gel mobility-shift assays demonstrate that the transcriptionally active 68-kDa fragment of the sterol regulatory element (SRE-1)-binding protein assays (SREBP-1) binds to an oligonucleotide containing the wild-type sequence but not to an oligonucleotide in which the CAC has been mutated. DNase 1 protection pattern (footprint) analysis indicates that SREBP-1 binds to nucleotides that include the CAC. Both the in vivo and in vitro assays are affected by mutagenesis of nucleotides adjacent to the CAC. Coexpression of SREBP with a wild-type FPP synthase promoter-reporter gene in CV-1 cells results in very high levels of reporter activity that is sterol-independent. In contrast, the reporter activity remained low when the promoter contained a mutation in the CAC trinucleotide. We conclude that sterol-regulated transcription of FPP synthase is controlled in part by the interaction of SREBP with a binding site that we have termed SRE-3. Identification of this element may prove useful in the identification of other genes that are both regulated by SREBP and involved in lipid biosynthesis.

Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia.

Productive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia lack activated cells as determined by an absence of staining for Ki-67 cell cycle antigen. The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells lack active NF-kappa B but express Sp1 as expected. DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication without ostensible immune stimulation.

Redefining the Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter and transcription initiation site in group I Burkitt lymphoma cell lines.

The Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter for the restricted Epstein-Barr virus (EBV) latency program operating in group I Burkitt lymphoma (BL) cell lines was previously identified incorrectly. Here we present evidence from RACE (rapid amplification of cDNA ends) cloning, reverse transcription-PCR, and S1 nuclease analyses, which demonstrates that the EBNA-1 gene promoter in group I BL cell lines is located in the viral BamHI Q fragment, immediately upstream of two low-affinity EBNA-1 binding sites. Transcripts initiated from this promoter, referred to as Qp, have the previously reported Q/U/K exon splicing pattern. Qp is active in group I BL cell lines but not in group III BL cell lines or in EBV immortalized B-lymphoblastoid cell lines. In addition, transient transfection of Qp-driven reporter constructs into both an EBV-negative BL cell line and a group I BL cell line gave rise to correctly initiated transcripts. Inspection of Qp revealed that it is a TATA-less promoter whose architecture is similar to the promoters of housekeeping genes, suggesting that Qp may be a default promoter which ensures EBNA-1 expression in cells that cannot run the full viral latency program. Elucidation of the genetic mechanism responsible for the EBNA-1-restricted program of EBV latency is an essential step in understanding control of viral latency in EBV-associated tumors.

A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system.

We have studied the functional consequences of a mutation in the epithelial Na+ channel that causes a heritable form of salt-sensitive hypertension, Liddle disease. This mutation, identified in the original kindred described by Liddle, introduces a premature stop codon in the channel beta subunit, resulting in a deletion of almost all of the C terminus of the encoded protein. Coexpression of the mutant beta subunit with wild-type alpha and gamma subunits in Xenopus laevis oocytes resulted in an approximately 3-fold increase in the macroscopic amiloride-sensitive Na+ current (INa) compared with the wild-type channel. This change in INa reflected an increase in the overall channel activity characterized by a higher number of active channels in membrane patches. The truncation mutation in the beta subunit of epithelial Na+ channel did not alter the biophysical and pharmacological properties of the channel--including unitary conductance, ion selectivity, or sensitivity to amiloride block. These results provide direct physiological evidence that Liddle disease is related to constitutive channel hyperactivity in the cell membrane. Deletions of the C-terminal end of the beta and gamma subunits of rat epithelial Na+ channel were functionally equivalent in increasing INa, suggesting that the cytoplasmic domain of the gamma subunit might be another molecular target for mutations responsible for salt-sensitive forms of hypertension.

Cannabinoid ligand-receptor signaling in the mouse uterus.

Using RNA (Northern) blot hybridization and reverse transcription-PCR, we demonstrate that the brain-type cannabinoid receptor (CB1-R) mRNA, but not the spleen-type cannabinoid receptor (CB2-R) mRNA, is expressed in the mouse uterus and that this organ has the capacity to synthesize the putative endogenous cannabinoid ligand, anandamide (arachidonylethanolamide). The psychoactive cannabinoid component of marijuana--delta 9-tetrahydrocannabinol (THC)--or anandamide, but not the inactive and nonpsychoactive cannabidiol (CBD), inhibited forskolin-stimulated cyclic AMP formation in the mouse uterus, which was prevented by pertussis toxin pretreatment. These results suggest that uterine CB1-R is coupled to inhibitory guanine nucleotide-binding protein and is biologically active. Autoradiographic studies identified ligand binding sites ([3H]anandamide) in the uterine epithelium and stromal cells, suggesting that these cells are perhaps the targets for cannabinoid action. Scatchard analysis of the binding of [3H]WIN 55212-2, another cannabinoid receptor ligand, showed a single class of high-affinity binding sites in the endometrium with an apparent Kd of 2.4 nM and Bmax of 5.4 x 10(9) molecules per mg of protein. The gene encoding lactoferrin is an estrogen-responsive gene in the mouse uterus that was rapidly and transiently up-regulated by THC, but not by CBD, in ovariectomized mice in the absence of ovarian steroids. This effect, unlike that of 17 beta-estradiol (E2), was not influenced by a pure antiestrogen, ICI 182780, suggesting that the THC-induced uterine lactoferrin gene expression does not involve estrogen receptors. We propose that the uterus is a new target for cannabinoid ligand-receptor signaling.