The development of pest resistance threatens the effectiveness of Bacillus thuringiensis (Bt) toxins used in transgenic and organic farming. Here, we demonstrate that (i) the major mechanism for Bt toxin resistance in Caenorhabditis elegans entails a loss of glycolipid carbohydrates; (ii) Bt toxin directly and specifically binds glycolipids; and (iii) this binding is carbohydrate-dependent and relevant for toxin action in vivo. These carbohydrates contain the arthroseries core conserved in insects and nematodes but lacking in vertebrates. We present evidence that insect glycolipids are also receptors for Bt toxin.
The association between nicotinic acetylcholine receptor (nAChR) dysfunction and cognitive decline in Alzheimer's disease (AD) has been widely exploited for its therapeutic potential. The effects of chronic nicotine exposure on A accumulation have been studied in both humans and animal models, but its therapeutic efficacy for AD neuropathology is still unresolved. To date, no in vivo studies have addressed the consequences of activating nAChRs on tau pathology. To determine the effects of chronic nicotine administration on A and tau pathology, we chronically administrated nicotine to a transgenic model of AD (3xTg-AD) in their drinking water. Here, we show that chronic nicotine intake causes an up-regulation of nicotinic receptors, which correlated with a marked increase in the aggregation and phosphorylation state of tau. These data show that nicotine exacerbates tau pathology in vivo. The increase in tau phosphorylation appears to be due to the activation of p38-mitogen-activated protein kinase, which is known to phosphorylate tau in vivo and in vitro. We also show that the 3xTg-AD mice have an age-dependent reduction of 7nAChRs compared with age-matched nontransgenic mice in specific brain regions. The reduction of 7nAChRs is first apparent at 6 months of age and is restricted to brain regions that show intraneuronal A42 accumulation. Finally, this study highlights the importance of testing compounds designed to ameliorate AD pathology in a model with both neuropathological lesions because of the differential effects it can have on either A or tau.
The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.
Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an 29.5-kDa noncovalent homodimer (dGal-1) that is widely expressed in many tissues. Here, we report that human recombinant dGal-1 bound preferentially and with high affinity (apparent Kd 2–4 µM) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Gal1–4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (–3Gal1–4GlcNAc1–)n) sequences, complex-type biantennary N-glycans, or novel chitin-derived glycans modified to contain terminal LN. Although terminal Gal residues are important for dGal-1 recognition, dGal-1 bound similarly to 3-sialylated and 2-fucosylated terminal LN, but not to 6-sialylated and 3-fucosylated terminal LN. The binding specificity of human recombinant dGal-1 was similar to that observed with purified bovine heart-derived dGal-1. Unexpectedly, dGal-1 bound free ligands in solution with relatively low affinity and displayed no preference for extended glycans, indicating that dGal-1 preferentially recognizes extended glycans only when they are surface-bound, such as found on cell surfaces. Human dGal-1 also bound to both native and desialylated human promyelocytic HL-60 cells with similar affinity as observed for immobilized long chain PL. Binding to these cells was reduced upon treatment with endo--galactosidase, which cleaves PL sequences, indicating that cell-surface PLs are ligands. To test the role of dimerization in dGal-1 binding, we examined the binding of a mutated form of dGal-1 that weakly dimerizes (monomeric Gal-1 (mGal-1)) and a covalently dimerized (chemically cross-linked) form of mGal-1 (cd-mGal-1). dGal-1 and cd-mGal-1 had similar affinities that were both 3.5-fold higher for immobilized PL than observed for mGal-1, suggesting that dGal-1 acts as a dimer to cross-link terminal LN units on immobilized PL. These results indicate that dGal-1 functions as a dimer to recognize LN units on extended PLs on cell surfaces.
P-selectin glycoprotein ligand-1 (PSGL-1) interactions with selectins regulate leukocyte migration in inflammatory lesions. In mice, selectin ligand activity regulating leukocyte recruitment and lymphocyte homing into lymph nodes results from the sum of unequal contributions of fucosyltransferase (FucT)-IV and FucT-VII, with FucT-VII playing a predominant role. Here we have examined the role of human FucT-IV and -VII in conferring L-selectin, P-selectin, and E-selectin binding activities to PSGL-1. Lewis x (Lex) carbohydrate was generated at the CHOdhfr– cell surface by FucT-IV expression, whereas sialyl Lex (sLex) was synthesized by FucT-VII. Both human FucT-IV and -VII had the ability to generate carbohydrate ligands that support L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a major role. Cooperation was observed between FucT-IV and -VII in recruiting L-, P-, or E-selectin-expressing cells on PSGL-1 and in regulating cell rolling velocity and stability. Additional rolling adhesion assays were performed to assess the role of Thr-57-linked core-2 O-glycans in supporting L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1. These studies confirmed that core-2 O-glycans attached to Thr-57 play a critical role in supporting L- and P-selectin-dependent rolling and revealed that additional binding sites support >75% of E-selectin-mediated rolling. The observations presented here indicate that human FucT-IV and -VII both contribute and cooperate in regulating L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a predominant role in conferring selectin binding activity to PSGL-1.
Several proteins linked to neurodegenerative diseases, such as the -amyloid precursor protein, amyloid -peptide, -secretase, and tau, undergo selective polarized sorting. We investigated polarized sorting of the mammalian prion protein (PrPC) and its homologue doppel (Dpl). In contrast to Dpl, which accumulates on the apical surface, PrPC is targeted selectively to the basolateral side in Madin-Darby canine kidney cells. An extensive deletion and domain swapping analysis revealed that the internal hydrophobic domain (HD) of PrP (amino acids 113–133) confers basolateral sorting in a dominant manner. PrP mutants lacking the HD are sorted apically, while Dpl chimeras containing the HD of PrP are directed to the basolateral membrane. Furthermore, a pathogenic PrP misse
The prosperity of an organism relies on its ability to respond to its ever changing environment. The mechanism for this adaptive response is simple in theory; external stimuli are received and integrated, thereby eliciting a concerted and appropriate response. This cellular communication depends largely on the transmission of signal couriers (i.e. "ligands") from one cell, which are then received via cell surface and intracellular recognition molecules (i.e. "receptors") on the recipient cell (1). The interaction of an activating ligand (i.e. "agonist") with the receptor ultimately results in transduction of the signal via a complex web of biochemical interactions to produce the required response.
The diversity of physiological responses that occur in multicellular organisms includes the modulation of the central and peripheral nervous system and cardiovascular, gastrointestinal, metabolic, reproductive, genitourinary, and immunological functions and reflects the number of ligand and receptor molecules that elicit them. These ligands can include environmental stimulants such as photons, odorants, tastants, pheromones, and viruses as well as native molecules, including various small molecule neurotransmitters, amino acids, polypeptides, hormones, nucleotides, ions, and lipids. To accommodate the selective recognition of these diverse ligands a number of large receptor superfamilies encompassing both membrane receptors (e.g. G protein-coupled receptors, receptor tyrosine kinases, ligand-gated ion channels, and integrins) and intracellular nuclear receptors have evolved (2). Taken in its entirety, that portion of the proteome dedicated to ligand reception has been described as the "receptorome" (3) and encompasses more than 5% of the human genome (2).
The insulin/insulin-like growth factor-like signaling pathway, present in all multicellular organisms, regulates diverse functions including growth, development, fecundity, metabolic homeostasis, and lifespan. In flies, ligands of the insulin/insulin-like growth factor-like signaling pathway, the Drosophila insulin-like peptides, regulate growth and hemolymph carbohydrate homeostasis during development and are expressed in a stage- and tissue-specific manner. Here, we show that ablation of Drosophila insulin-like peptide-producing median neurosecretory cells in the brain leads to increased fasting glucose levels in the hemolymph of adults similar to that found in diabetic mammals. They also exhibit increased storage of lipid and carbohydrate, reduced fecundity, and reduced tolerance of heat and cold. However, the ablated flies show an extension of median and maximal lifespan and increased resistance to oxidative stress and starvation.
Alternative pre-mRNA splicing affects a majority of human genes and plays important roles in development and disease. Alternative splicing (AS) events conserved since the divergence of human and mouse are likely of primary biological importance, but relatively few of such events are known. Here we describe sequence features that distinguish exons subject to evolutionarily conserved AS, which we call alternative conserved exons (ACEs), from other orthologous human/mouse exons and integrate these features into an exon classification algorithm, ACESCAN. Genome-wide analysis of annotated orthologous human-mouse exon pairs identified 2,000 predicted ACEs. Alternative splicing was verified in both human and mouse tissues by using an RT-PCR-sequencing protocol for 21 of 30 (70%) predicted ACEs tested, supporting the validity of a majority of ACESCAN predictions. By contrast, AS was observed in mouse tissues for only 2 of 15 (13%) tested exons that had EST or cDNA evidence of AS in human but were not predicted ACEs, and AS was never observed for 11 negative control exons in human or mouse tissues. Predicted ACEs were much more likely to preserve the reading frame and less likely to disrupt protein domains than other AS events and were enriched in genes expressed in the brain and in genes involved in transcriptional regulation, RNA processing, and development. Our results also imply that the vast majority of AS events represented in the human EST database are not conserved in mouse.
Phosphomannomutase (PMM) deficiency causes Congenital Disorder of Glycosylation (CDG)-Ia, a broad-spectrum disorder with developmental and neurological abnormalities. PMM converts mannose-6-P (M6P) to mannose-1-P, a precursor of GDP-mannose used to make Glc3Man9GlcNAc2-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase (OT) for protein N-linked glycosylation. Hepatically-produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [3H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [3H]LLO intermediates. As these are thought to be poor OT substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, fluorophore-assisted carbohydrate electrophoresis (FACE, a non-radioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [3H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc3Man9GlcNAc2-P-P-dolichol, without hypoglycosylation, in CDG-Ia fibroblasts grown with physiological glucose. This suggested a “missing link” to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, M6P was a specific activator for cleavage of Glc3Man9GlcNAc2-P-P-dolichol. This led to futile cycling of the LLO pathway, exacerbated by GDP-mannose/PMM deficiency. The possibilities that M6P may accumulate in hepatocytes, and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy, with CDG-Ia patients are discussed.
During transcription initiation RNA polymerases retain interactions with their promoters until the RNA is extended to 8-13 nts, at which point the polymerase releases the promoter and moves downstream. It has been shown that release of the T7 promoter is inhibited if the T7 RNA polymerase:promoter interaction is strengthened. Here we ask if release will be facilitated if the T7 promoter:polymerase interaction is weakened by the introduction of promoter mutations known to reduce promoter activity. Using chemical and enzymatic probes to monitor the position of the polymerase as a function of RNA length we find that promoter mutations upstream of -4 facilitate release of the polymerase from the promoter, but more downstream mutations do not have such effects. We also find that released complexes turn over more slowly than promoter bound complexes, indicating that retention of promoter interactions contributes to the dissociation of short RNAs during initial transcription.
Until relatively recently, RNA has taken a predominantly backstage role compared to protein in genome studies. However, this is changing dramatically with the discovery of a plethora of RNAs that do not act as messenger (mRNA), transfer (tRNA), or ribosomal (rRNA) RNAs (1–3). These noncoding RNAs (ncRNAs) play a role in a variety of processes such as transcriptional regulation, chromosome replication, RNA processing and modification, and protein degradation and translocation. Even so, ncRNAs usually lack the statistical signals in their primary sequence (like ORFs and codon bias) that have been used to such great effect in the identification of novel protein encoding genes, making the task of systematically identifying new ncRNAs in genomes currently one of the most exciting challenges in computational biology. The work of Washietl et al. in this issue of PNAS (4) faces this challenge head on. Through an elegant use of structural properties of RNA, the authors present an efficient comparative genomics approach to identifying novel ncRNAs and related genomic elements that promises to significantly contribute to the burgeoning field of computational RNomics.
Pig embryonic tissues represent an attractive option for organ transplantation. However, the achievement of optimal organogenesis after transplantation, namely, maximal organ growth and function without teratoma development, represents a major challenge. In this study, we determined distinct gestational time windows for the growth of pig embryonic liver, pancreas, and lung precursors. Transplantation of embryonic-tissue precursors at various gestational ages [from E (embryonic day) 21 to E100] revealed a unique pattern of growth and differentiation for each embryonic organ. Maximal liver growth and function were achieved at the earliest teratoma-free gestational age (E28), whereas the growth and functional potential of the pancreas gradually increased toward E42 and E56 followed by a marked decline in insulin-secreting capacity at E80 and E100. Development of mature lung tissue containing essential respiratory system elements was observed at a relatively late gestational age (E56). These findings, showing distinct, optimal gestational time windows for transplantation of embryonic pig liver, pancreas, and lung, might explain, in part, the disappointing results in previous transplantation trials and could help enhance the chances for successful implementation of embryonic pig tissue in the treatment of a wide spectrum of human diseases.
Contemporary climate change is characterized both by increasing mean temperature and increasing climate variability such as heat waves, storms, and floods. How populations and communities cope with such climatic extremes is a question central to contemporary ecology and biodiversity conservation. Previous work has shown that species diversity can affect ecosystem functioning and resilience. Here, we show that genotypic diversity can replace the role of species diversity in a species-poor coastal ecosystem, and it may buffer against extreme climatic events. In a manipulative field experiment, increasing the genotypic diversity of the cosmopolitan seagrass Zostera marina enhanced biomass production, plant density, and faunal abundance, despite near-lethal water temperatures due to extreme warming across Europe. Net biodiversity effects were explained by genotypic complementarity rather than by selection of particularly robust genotypes. Positive effects on invertebrate fauna suggest that genetic diversity has second-order effects reaching higher trophic levels. Our results highlight the importance of maintaining genetic as well as species diversity to enhance ecosystem resilience in a world of increasing uncertainty.
CYP7B1 is the enzyme responsible for hydroxylation and termination of the estrogenic actions of the androgen metabolite, 5-androstane-3, 17-diol (3Adiol). 3Adiol is estrogenic in ER or ER positive cells only if they do not express CYP7B1. In this study we show that female CYP7B1-/- mice experience early onset of growth of the uterus and mammary glands and commence estrus cycles 2 days earlier than their wild-type littermates. Adult mammary glands and uteri appear to be under continuous estrogenic stimulation. We conclude that, by cell-specific regulation of the estrogenicity of 3Adiol, CYP7B1 performs two major tasks: (i) it allows 3Adiol to have growth inhibitory effects through ER and (ii) it permits estradiol-specific activation of estrogen receptors by protection of certain cells from the estrogenic effects of 3Adiol. When CYP7B1 is inactivated, 3Adiol activates estrogen receptors indiscriminately, and the overall effect is prolonged and inappropriate exposure to estrogen.
A targeted increase in mitochondrial uncoupling in adult neurons reduced the generation of reactive oxygen species (ROS) and extended life span without diminishing critical physiological systems in the fly. This defines a new point for life span intervention, modifying the metabolic efficiency of the mitochondria. The successful initiation of this intervention during adult life in the fly suggests that selective alterations in mitochondrial physiology could make a positive difference in health and longevity for adults of other species.
Almost always, obesity is an obligate prerequisite for the development of type 2 diabetes mellitus. Loss of a well-known lipid phosphatase appears to prevent insulin-resistant diabetes in mice by removal of a positive regulator of adiposity.
A transcription factor cascade drives adipogenesis. Two new members of this cascade have been uncovered—Kruppel-like factor 5 (KLF5), which is induced by C/EBPÉ¿ and ɬ and acts in concert with C/EBPÉø, É¿, and ɬ to activate PPARÉ¡2 expression, and Krox20, which is upstream of C/EBPÉ¿ expression and one of the earliest factors induced during adipogenesis.
We explore the hypothesis that pathology of Huntington's disease involves multiple cellular mechanisms whose contributions to disease are incrementally additive or synergistic. We provide evidence that the photoreceptor neuron degeneration seen in flies expressing mutant human huntingtin correlates with widespread degenerative events in the Drosophila CNS. We use a Drosophila Huntington's disease model to establish dose regimens and protocols to assess the effectiveness of drug combinations used at low threshold concentrations. These proof of principle studies identify at least two potential combinatorial treatment options and illustrate a rapid and cost-effective paradigm for testing and optimizing combinatorial drug therapies while reducing side effects for patients with neurodegenerative disease. The potential for using prescreening in Drosophila to inform combinatorial therapies that are most likely to be effective for testing in mammals is discussed.
To investigate the biological significance of a longevity mutation found in daf-2 of Caenorhabditis elegans, we generated a homologous murine model by replacing Pro-1195 of insulin receptors with Leu using a targeted knock-in strategy. Homozygous mice died in the neonatal stage from diabetic ketoacidosis while heterozygous mice showed the suppressed kinase activity of the insulin receptor, but grew normally without spontaneously developing diabetes during adulthood. We examined heterozygous insulin receptor mutant mice for longevity phenotypes. Under 80% oxygen, mutant female mice survived 33.3% longer than wild-type female mice while mutant male mice survived 18.2% longer than wild-type male mice. These results suggested that mutant mice acquired more resistance to oxidative stress, but the benefit of the longevity mutation was more pronounced in females than males. Manganese superoxide dismutase activity in mutant mice was significantly up-regulated, suggesting that the suppressed insulin signaling leads to an enhanced antioxidant defense. To analyze the molecular basis of the gender difference, we administered estrogen to mutant mice. It was found that the survival of mice under 80% oxygen was extended when they were administered estradiol. In contrast, mutant and wild-type female mice showed shortened survivals when their ovaries were removed. The influence of estrogen is remarkable in mutant mice compared to wild-type mice, suggesting that estrogen modulates insulin signaling in mutant mice. Furthermore, we showed additional extension of survival under oxidative conditions when their diet was restricted. Collectively, we show that 3 distinct signals; insulin, estrogen, and dietary signals, work in independent and cooperative ways to enhance the resistance to oxidative stress in mice.
Cyanobacteria are among the simplest organisms that show daily rhythmicity. Their circadian rhythms consist of the localization, interaction, and accumulation of various proteins, including KaiA, KaiB, KaiC and SasA. We have determined the 1.9 Å resolution crystallographic structure of the cyanobacterial KaiB clock protein from Synechocystis sp. PCC 6803. This homotetrameric structure reveals a novel KaiB interface for protein-protein interaction; the protruding hydrophobic helix-turn-helix motif of one subunit fits into a groove between two ?-strands of the adjacent subunit. A cyanobacterial mutant, in which the Asp-Lys salt bridge mediating this tetramer-forming interaction is disrupted by mutation of Asp to Gly, exhibits severely impaired rhythmicity (a short free-running period; ~19 hours). The KaiB tetramer forms an open square, with positively charged residues around the perimeter. KaiB is localized on the phospholipid-rich membrane and translocates to the cytosol to interact with the other Kai components, KaiA and KaiC. Kai B antagonizes the action of KaiA on KaiC, and shares a sequence-homologous domain with the SasA kinase. Based on our structure, we discuss functional roles for KaiB in the circadian clock.
Darwinian theory holds that natural selection operating on randomly generated chemical structures is the only mechanism to create biomolecules that confer fitness upon their hosts. A considerable gap separates this biological truism from experimental reality, however. As Kreitman and Akashi (1995) noted a decade ago, making the connection between molecular behavior and fitness has proven to be remarkably difficult.
The OMEGA investigation, on board the ESA/Mars Express mission, is mapping the surface composition of Mars, at a 0.3 to 5 km resolution, by means of VIS-NIR hyperspectral reflectance imagery. The data acquired during the first nine months of the mission already reveal a diverse and complex surface mineralogy, offering key insights to the evolution of Mars. OMEGA has identified and mapped mafic iron-bearing silicates of both the northern and southern crust; localized concentrations of hydrated phyllosilicates and sulfates, and no carbonates; ices and frosts, with a water-ice composition of the north polar perennial cap, as for the south cap, covered by a thin CO2 - ice veneer.
How specialized are the separable neural pathways that support perception? A long debate has concerned whether neurons that encode color also encode aspects of shape, such as orientation. In this paper, Engel shows that the color pathways include large numbers of unoriented and oriented neurons and that both these populations are important for color perception. Thus, the color pathways also encode information about form, but this does not make them less specialized for color perception.
Memory storage is often modeled as being associated with a permanent switch-like transition in the state of a set of synapses. For ongoing storage of everyday occurrences, such a mechanism is prone to rapid forgetting due to the overwriting of previously stored memories by new ones. In this modeling study, Fusi et al. construct and study a model in which each synapse has a cascade of states with different levels of plasticity, connected by metaplastic transistons. A synaptic plasticity mechanism that involves a cascade of transitions with a wide range of degrees of plasticity and lifetimes performs much better than a switch-like model. This suggests that the variety and complexity in forms of activity-dependent synaptic plasticity may be an essential element, rather than a complicating factor, in memory storage.
In vertebrate olfactory receptor neurons (ORNs), the odorant-triggered receptor current is mainly carried by Cl-efflux through a Ca2+-activated anion channel, and it crucially depends on a high intracellular Cl-concentration. Reisert et al. show that a Na+-K+-2Cl-cotransporter (NKCC1) is required for this Cl-current, by maintaining a driving force for Cl-efflux. Surprisingly, immunocytochemistry indicates that NKCC1 is located on the somata and dendrites of ORNs rather than the cilia, where transduction occurs. This topography is remarkably similar to the situation in secretory epithelial cells, where Cl-uptake at the basolateral membrane and Cl-efflux at the apical membrane facilitate transepithelial fluid movement.The context and implications of this work are discussed in a Preview by Restrepo.
Until recently, our knowledge of neurotransmitter receptor biology came from anatomical, electrophysiological, and biochemical studies. Genome and cDNA sequencing projects have identified numerous receptor subtypes with distinct ligand binding properties. For example, 18 genes encode glutamate receptor ion channels. In this paper, Mark Mayer presents crystal structures of GluR5 and GluR6, two “kainate receptor” genes with close to 90% amino acid sequence identity, and reveals how unique functional properties are achieved via subtle changes in amino acid sequence. The results of such studies open the door for the development of subtype-specific ligands and are likely to play an emergent role in neurotransmitter receptor biology in the next decade.
Genetic information can be altered through the enzymatic modification of nucleotide sequences. This process, known as editing, was originally identified in the mitochondrial RNA of trypanosomes and later found to condition events as diverse as neurotransmission and lipid metabolism in mammals. Recent evidence reveals that editing enzymes may fulfill one of their most essential roles in the defense against infectious agents: first, as the mediators of antibody diversification, a step crucial for building adaptive immunity, and second, as potent intracellular poisons for the replication of viruses. Exciting questions are raised, which take us to the depth of the intimate relations between vertebrates and the microbial underworld.
Prions typically accumulate in nervous and lymphoid tissues. Because proinflammatory cytokines and immune cells are required for lymphoid prion replication, we tested whether inflammatory conditions affect prion pathogenesis. We administered prions to mice with five inflammatory diseases of the kidney, pancreas, or liver. In all cases, chronic lymphocytic inflammation enabled prion accumulation in otherwise prion-free organs. Inflammatory foci consistently correlated with lymphotoxin up-regulation and ectopic induction of FDC-M1+ cells expressing the normal cellular prion protein PrPC. By contrast, inflamed organs of mice lacking lymphotoxin- or its receptor did not accumulate the abnormal isoform PrPSc, nor did they display infectivity upon prion inoculation. By expanding the tissue distribution of prions, chronic inflammatory conditions may act as modifiers of natural and iatrogenic prion transmission.
The sex pheromone of the German cockroach, Blattella germanica, has been characterized as gentisyl quinone isovalerate. This cockroach is a major cause of allergic disease and serves as a mechanical vector of pathogens, making it one of the most important residential and food-associated pests worldwide. The sex pheromone–producing gland in adult females was identified in 1993, but thermal instability of the pheromone made characterization difficult. Now, using a new preparative gas chromatography approach coupled with electroantennographic detection, we have isolated and characterized the pheromone, which we term blattellaquinone, and confirmed the identification by chemical synthesis. The synthetic pheromone was active in behavioral assays and highly effective in field trapping tests, which suggest that it may provide a new tool in cockroach population detection, monitoring, and control.
Individual differences in DNA sequence are the genetic basis of human variability. We have characterized whole-genome patterns of common human DNA variation by genotyping 1,586,383 single-nucleotide polymorphisms (SNPs) in 71 Americans of European, African, and Asian ancestry. Our results indicate that these SNPs capture most common genetic variation as a result of linkage disequilibrium, the correlation among common SNP alleles. We observe a strong correlation between extended regions of linkage disequilibrium and functional genomic elements. Our data provide a tool for exploring many questions that remain regarding the causal role of common human DNA variation in complex human traits and for investigating the nature of genetic variation within and between human populations.
To study the mechanism of gel-forming mucin packaging within mucin granules, we generated human mucous/goblet cells stably expressing a recombinant MUC5AC domain fused to green fluorescent protein (GFP). The fusion protein, named SHGFP-MUC5AC/CK, was accumulated within the granules together with the native MUC5AC. Inhibition of protein synthesis or the disorganization of the Golgi complex did not result in diminished SHGFP-MUC5AC/CK intragranular signals, consistent with long-term storage of the fusion protein. However, SHGFP-MUC5AC/CK was rapidly discharged from the granules upon incubation of the cells with ATP, an established mucin secretagogue. Several criteria indicated SHGFP-MUC5AC/CK was not covalently linked to endogenous MUC5AC. FRAP analysis suggested that the SHGFP-MUC5AC/CK intragranular mobile fraction (Mf) and mobility, respectively, were significantly smaller than in the endoplasmic reticulum lumen. Incubation of the cells with bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase, did not alter the fusion protein mobility, although it significantly increased (~20%) intragranular SHGFP-MUC5AC/CK Mf. In addition, the granules in bafilomycin-incubated cells typically exhibited a heterogeneous intralumenal distribution of the fluorescent fusion protein. These results are consistent with a model of the mucin granule intralumenal organization with two phases: a) a mobile phase, in which secretory proteins diffuse as in the endoplasmic reticulum lumen but at a lower rate; and b) an immobile phase or matrix in which proteins are immobilized by non-covalent, pH-dependent interactions. An intralumenal acidic pH, maintained by the vacuolar H+-ATPase, is one of the critical factors for secretory protein binding to the immobile phase and also for its organization.
The androgen receptor (AR) activates target gene expression in the presence of agonist ligands via the recruitment of transcriptional coactivators, but recent work shows that overexpression of the nuclear corepressors NCoR and SMRT attenuates this agonist-mediated AR activation. Here we demonstrate using NCoR siRNA and chromatin immunoprecipitation that endogenous NCoR is recruited to and represses the dihydrotestosterone (DHT)-liganded AR. Furthermore this study shows that NCoR and coactivators compete for AR in the presence of DHT. AR antagonists such as bicalutamide that are currently in use for prostate cancer treatment can also mediate NCoR recruitment, but mifepristone (RU486) at nanomolar concentrations is unique in its ability to markedly enhance the AR-NCoR interaction. The RU486-liganded AR interacted with a C-terminal fragment of NCoR, and this interaction was mediated by the two most C-terminal nuclear receptor interacting domains (RIDs) present in NCoR. Significantly, in addition to the AR ligand binding domain, the AR N terminus was also required for this interaction. Mutagenesis studies demonstrate that the N-terminal surface of the AR-mediating NCoR recruitment was distinct from tau5 and from the FXXLF motif that mediates agonist-induced N-C-terminal interaction. Taken together these data demonstrate that NCoR is a physiological regulator of the AR and reveal a new mechanism for AR antagonism that may be exploited for the development of more potent AR antagonists.
Nicotine, a major component in tobacco, has been implicated as a potential factor that promotes the development of lung cancer. However, the molecular mechanism of its action is still unclear. In this study, we have shown that, via nicotinic acetylcholine receptors, persistent exposure of mouse epithelial cells to nicotine elicits Ras signaling and subsequent Raf/MAP kinase activity, accompanied by a significant increase in cyclin D1 promoter activity and its protein expression. AP-1 is required for activation of the cyclin D1 promoter. The induction of cyclin D1 expression and its promoter activity by nicotine is abolished by the suppression of Raf/MAP kinase signaling. Furthermore, upon nicotine treatment, the cells do not arrest in the G1 phase of the cell cycle following serum starvation. The perturbation of the G1 cell cycle checkpoint is caused by the deregulation of retinoblastoma/E2F activity. Therefore, our data indicated that by targeting the Ras pathway, long-term exposure to nicotine disrupts cell cycle restriction machinery and thus potentiates tumor development.
Several signaling pathways that monitor the dynamic state of the cell converge on the tumor suppressor p53. The ability of p53 to process these signals and exert a dynamic downstream response in the form of cell cycle arrest and/or apoptosis is crucial for preventing tumor development. This p53 function is abrogated by p53 gene mutations leading to alteration of protein conformation. Hsp90 has been implicated in regulating both wild-type and mutant p53 conformations, and Hsp90 antagonists are effective for the therapy of some human tumors. Using cell lines that contain human tumor-derived temperature-sensitive p53 mutants we show that Hsp90 is required for both stabilization and reactivation of mutated p53 at the permissive temperature. A temperature decrease to 32 °C causes conversion to a protein conformation that is capable of inducing expression of MDM2, leading to reduction of reactivated p53 levels by negative feedback. Mutant reactivation is enhanced by simultaneous treatment with agents that stabilize the reactivated protein and is blocked by geldanamycin, a specific inhibitor of Hsp90 activity, indicating that Hsp90 antagonist therapy and therapies that act to reactivate mutant p53 will be incompatible. In contrast, Hsp90 is not required for maintaining wild-type p53 or for stabilizing wild-type p53 after treatment with chemotherapeutic agents, indicating that Hsp90 therapy might synergize with conventional therapies in patients with wild-type p53. Our data demonstrate the importance of the precise characterization of the interaction between p53 mutants and stress proteins, which may shed valuable information for fighting cancer via the p53 tumor suppressor pathway.
Insulin inhibits glucagon gene transcription, and insulin deficiency is associated with hyperglucagonemia that contributes to hyperglycemia in diabetes mellitus. However, the insulin signaling pathway to the glucagon gene is unknown. Protein kinase B (PKB) is a key regulator of insulin signaling and glucose homeostasis. Impaired PKB function leads to insulin resistance and diabetes mellitus. Therefore, the role of PKB in the regulation of glucagon gene transcription was investigated. After transient transfections of glucagon promoter-reporter genes into a glucagon-producing islet cell line, the use of kinase inhibitors indicated that the inhibition of glucagon gene transcription by insulin depends on phosphatidylinositol (PI) 3-kinase. Furthermore, insulin caused a PI 3-kinase-dependent phosphorylation and activation of PKB in this cell line as revealed by phospho-immunoblotting and kinase assays. Overexpression of constitutively active PKB mimicked the effect of insulin on glucagon gene transcription. Both insulin and PKB responsiveness of the glucagon promoter were abolished when the binding sites for the transcription factor Pax6 within the G1 and G3 promoter elements were mutated. Recruitment of Pax6 or its potential coactivator, the CREB-binding protein (CBP), to G1 and G3 by using the GAL4 system restored both insulin and PKB responsiveness. These data suggest that insulin inhibits glucagon gene transcription by signaling via PI 3-kinase and PKB, with the transcription factor Pax6 and its potential coactivator CBP being critical components of the targeted promoter-specific nucleoprotein complex. The present data emphasize the importance of PKB in insulin signaling and glucose homeostasis by defining the glucagon gene as a novel target gene for PKB.
In eukaryotic cells, initiation of DNA replication is achieved by an ordered assembly of protein complexes at origins of replication. This process consists of two steps, licensing of origins in late M or early G1 phase and firing of origins at different times during S phase. Origin licensing is mediated by the assembly of pre-replicative complexes (pre-RCs) on origins. At the center of the pre-RC is the origin recognition complex (ORC), a six-subunit complex (Orc1–Orc6), which binds origins of replication (2). During late M and early G1 phase, Cdt1 and Cdc6 bind origins in an ORC-dependent manner and then cooperatively recruit a putative helicase, Mcm2–7 complex, to origins (2) (Fig. 1). Once pre-RCs are assembled, origins are licensed for replication in the subsequent S phase and ready to fire. A key event for origin firing, the second step of the initiation process, is loading of Cdc45 on origins (Fig. 1). This step is triggered by the action of two kinases, cyclin-Cdk and Cdc7-Dbf4. Although cyclin-Cdk complex works as a global activator for S phase, Cdc7-Dbf4 acts as an activator in the initiation step at the individual origins. Following Cdc45 loading on origins, the single-stranded DNA (ssDNA)-binding protein, RPA, and the primase-DNA polymerase complex are loaded on chromatin to initiate DNA replication (2).
Karl Paul Link (1901–1978) received his Ph.D. in 1925 from the University of Wisconsin, working with plant biochemist William E. Tottingham. He spent the next 2 years in Europe studying carbohydrate chemistry with Sir James Irvine in Scotland, microchemistry with Fritz Pregl in Austria, and organic chemistry with Paul Karrer in Switzerland. He returned to the University of Wisconsin as an assistant professor in agricultural chemistry (now biochemistry) in 1927 and was promoted to associate professor in 1928.
Initially when he set up his laboratory, Link concentrated on plant carbohydrates and soon established himself as one of the outstanding carbohydrate chemists of his day. Using the microchemical techniques he learned with Pregl, he and his students were able to characterize carbohydrate derivatives that they had isolated and synthesized.
However, the direction of Link's research changed drastically when he became involved in the isolation and characterization of the hemorrhagic factor produced in spoiled sweet clover hay. These experiments are the subject of the three Journal of Biological Chemistry (JBC) Classics reprinted here. Sweet clover was widely used as hay in the 1920s when a series of wet summers had led to an epidemic of "bleeding disease" in cattle. The cause of the disease was traced to sweet clover hay that had been improperly cured and infected with molds. There was also evidence that the defective coagulation in the cows was due to a deficiency in prothrombin.
Isoprenylcysteine carboxyl methyltransferase (Icmt) is an endoplasmic reticulum enzyme responsible for carboxylmethylation of prenylated proteins. This methylation is critical for the proper localization and functioning of Ras proteins, thus making it a potentially useful chemotherapeutic target for Ras-based cancers. However, Icmts have multiple membrane spanning domains, which present a challenge for their purification and characterization. In this paper, Jessica L. Anderson and colleagues purify an Icmt from S. cerevisiae (Ste14p) to homogeneity using high level expression of the His-tagged protein followed by solubilization in -D-dodecylmaltoside and metal affinity column chromatography. The purified enzyme retained activity and was able to methylate both a model substrate (N-acetyl-S-farnesyl-L-cysteine) and Ras. The authors also resolved the question as to whether the enzyme prefers farnesylated or geranylgeranylated substrates: it likes them both equally. These results pave the way for further characterization of this carboxyl methyltransferase as well as purification of other integral membrane proteins.
We aimed at understanding molecular events involved in the activation of a member of the G protein coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue N7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of TSHr activation. N7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues T6.43 and D6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of ~14 and ~10 relative to wt TSHr, respectively. Upon receptor activation N7.49 adopts the trans conformation to interact with D2.50 and a putatively charged residue that remains to be identified, as shown by the fact that D2.50A, D2.50N, D2.50N/N7.49D, and N7.49D mutants can all be activated by TSH. In addition, the conserved L2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state since the L2.46A mutation increases constitutive activity by a factor of ~13 relative to wt TSHr. As residues L2.46, D2.50, and N7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.
Signaling by Drosophila Toll pathway activates two Rel/NF-kappaB transcription factors—Dorsal (Dl) and Dorsal-related immune factor (Dif). Dl plays a central role in the establishment of dorso-ventral polarity during early embryogenesis, whereas Dif mediates the Toll receptor-dependent antifungal immune response in adult Drosophila. The absence of a Dif ortholog in mosquito genomes suggests that Dl may play its functional role in the mosquito Toll-mediated innate immune responses. We have cloned and molecularly characterized the gene homologous to Drosophila Dl and to Anopheles gambiae REL1 (Gambif1) from the yellow fever mosquito Aedes aegypti, named AaREL1. AaREL1 alternative transcripts encode two isoforms, AaREL1-A and AaREL1-B. Both transcripts are enriched during embryogenesis and are inducible by septic injury in larval and female mosquitoes. AaREL1 and AaREL2 (Aedes Relish) selectively bind to different B motifs from insect immune gene promoters. Ectopic expression of AaREL1-A in both Drosophila mbn-2 cells and transgenic flies specifically activates Drosomycin and results in increased resistance against the fungus Beauveria bassiana. AaREL1-B acted cooperatively with AaREL1-A to enhance the immune gene activation in Aag-2 cells. The RNAi knockouts revealed that AaREL1 affected the expression of Aedes homologue of Drosophila Serpin-27A and mediated specific antifungal immune response against B. bassiana. These results indicate that the homologue of Dl, but not that of Dif, is a key regulator of the Toll antifungal immune pathway in Ae. aegypti female mosquitoes.
Intestinal fatty acid binding protein (I-FABP) is a small protein which binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than that necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen ~ bezafibrate > S-ibuprofen >> nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds, however, the freely water soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity, however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.
The E2F transcription factor family plays a crucial and well-established role in cell cycle progression. Deregulation of E2F activities in vivo leads to developmental defects and cancer. Based on current evidence in the field, mammalian E2Fs can be functionally categorized into either transcriptional activators (E2F1, E2F2 and E2F3a) or repressors (E2F3b, E2F4, E2F5, E2F6 and E2F7). We have identified a novel E2F family member, E2F8, which is conserved in mice and humans and has its counterpart in Arabidopsis thaliana (E2Ls). Interestingly, E2F7 and E2F8 share unique structural features that distinguish them from other mammalian E2F repressor members, including the presence of two distinct DNA-binding domains and the absence of DP-dimerization, Retinoblastoma-binding, and transcriptional activation domains. Like E2F7, over-expression of E2F8 significantly slows down the proliferation of primary mouse embryonic fibroblasts. These observations, together with the fact that E2F7 and E2F8 can homodimerize and are expressed in the same adult tissues, suggest that they may have overlapping and perhaps synergistic roles in the control of cellular proliferation.
The flagellum of Methanococcus voltae is composed of 4 structural flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3. These proteins possess a total of 15 potential N-linked sequons (N-X-S/T) and show a mass shift on SDS-PAGE indicating significant post-translational modification. We describe here the structural characterization of the flagellin glycan from M. voltae using mass spectrometry to examine the proteolytic digests of the flagellin proteins in combination with NMR analysis of the purified glycan using a sensitive,cryogenically cooled probe. NanoLC-MS/MS analysis of the proteolytic digests of the flagellin proteins revealed that they are post-translationally modified with a novel N-linked trisaccharide of mass 779 Da which is composed of three sugar residues with masses of 318 Da, 258 Da and 203 Da, respectively. In every instance the glycan is attached to the peptide through the asparagine residue of a typical N-linked sequon. The glycan modification has been observed on 14 of the 15 sequon sites present on the four flagellin structural proteins. The novel glycan structure elucidated by NMR analysis was shown to be a trisaccharide composed of beta-ManpNAcA6Thr-(1-4)-beta-GlcpNAc3NAcA-(1-3)-beta-GlcpNAc linked to Asn. In addition, the same trisaccharide was identified on a tryptic peptide of the S-layer protein from this organism implicating a common N-linked glycosylation pathway.
Large-scale quantitative analysis of transcriptional co-expression has been used to dissect regulatory networks and to predict the functions of new genes discovered by genome sequencing in model organisms such as yeast. Although the idea that tissue-specific expression is indicative of gene function in mammals is widely accepted, it has not been objectively tested nor compared with the related but distinct strategy of correlating gene co-expression as a means to predict gene function.
The phosphoprotein Dishevelled (Dsh) is an essential component of Wnt signaling pathways and transduces signals into three separate branches, the canonical, non-canonical and Ca2+ pathways. How Dsh focuses signaling into these branches remains mysterious, but a new study reveals the importance of nuclear localization of Dsh for pathway-specific activation.
The steroid hormone estrogen regulates many functionally unrelated processes in numerous tissues. Although it is traditionally thought to control transcriptional activation through the classical nuclear estrogen receptors, it also initiates many rapid nongenomic signaling events. We found that of all G protein-coupled receptors characterized to date, GPR30 is uniquely localized to the endoplasmic reticulum, where it specifically binds estrogen and fluorescent estrogen derivatives. Activating GPR30 by estrogen resulted in intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus. Thus, GPR30 represents an intracellular transmembrane estrogen receptor that may contribute to normal estrogen physiology as well as pathophysiology.
We show that the specific subcellular distribution of H- and Nras GTPases is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi. Depalmitoylation redistributes farnesylated Ras in all membranes followed by repalmitoylation and trapping at the Golgi from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalisation. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras-GTP. Different de/repalmitoylation kinetics account for isoform-specific activation responses in response to growth factors.
The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitantly with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. We show that both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. Our results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane.
Murine ES cells can be maintained as a pluripotent, self-renewing population by LIF/STAT3-dependent signaling. The downstream effectors of this pathway have not been previously defined. In this report, we identify a key target of the LIF self-renewal pathway by showing that STAT3 directly regulates the expression of the Myc transcription factor. Murine ES cells express elevated levels of Myc and following LIF withdrawal, Myc mRNA levels collapse and Myc protein becomes phosphorylated on threonine 58 (T58), triggering its GSK3ß dependent degradation. Maintained expression of stable Myc (T58A) renders self-renewal and maintenance of pluripotency independent of LIF. By contrast, expression of a dominant negative form of Myc antagonizes self-renewal and promotes differentiation. Transcriptional control by STAT3 and suppression of T58 phosphorylation are crucial for regulation of Myc activity in ES cells and therefore in promoting self-renewal. Together, our results establish a mechanism for how LIF and STAT3 regulate ES cell self-renewal and pluripotency.
It is generally thought that regenerating and transdetermining cells regress from a mature, committed state to an embryonic-like “younger” state, thereby increasing their developmental potential. By examining the cell cycle and growth characteristics of multipotent regenerating and transdetermining Drosophila leg imaginal disc cells, Sustar and Schubiger challenge this view. The authors document that regenerating cells progress directly from their current developmental state to a new one, without reverting to a “younger” cell cycle. By contrast, cells undergoing transdetermination are subject to transient and unique cell-cycle and cell-size changes, suggesting the existence of a novel, stem cell-like state, which is neither embryonic nor mature.
Vertebrate nuclear receptors SF-1 and LRH-1 regulate crucial aspects of development, endocrine homeostasis, and metabolism. Both have been categorized as orphan receptors since their corresponding ligands have thus far not been identified. Here Krylova et al. present structural analyses revealing that these receptors bind phospholilpids. Further studies show that phosphatidyl inositol phosphates are candidate ligands and are required for optimal receptor activity. Intriguingly, evolutionary analysis indicates that ligand binding is the ancestral state of these receptors but was uniquely diminished or altered in the rodent LRH-1 lineage. These results identify SF-1 and LRH-1 as novel mediators of phospholipid signaling.
Despite decades of investigation, the mechanisms that control faithful segregation of bacterial chromosomes have remained elusive. Here, Gitai et al. use a combination of pharmacological and biochemical approaches to demonstrate a direct role for the actin-like bacterial cytoskeleton protein MreB. The findings suggest a two-step model for chromosome segregation in Caulobacter crescentus: MreB is essential for segregation of the region of the chromosome nearest the origin of replication, while the rest of the chromosome follows through an MreB-independent mechanism. The use of cytoskeletal elements for bacterial chromosome segregation raises the exciting possibility that bacterial and eukaryotic mitosis share more in common than was ever appreciated.
The ability of a cancer cell to invade surrounding tissue and to metastasize is governed by complex interactions between the tumor and its microenvironment. PAR1 is a protease-activated G protein-coupled receptor proposed to be involved in the invasive and metastatic properties of various cancers. However, the protease responsible for activating the proinvasive functions of PAR1 remains to be identified. Here Boire et al. discover that a matrix metalloprotease, MMP-1, directly cleaves and activates PAR1 resulting in cancer cell invasion and tumorigenesis. Blocking the MMP1-PAR1 pathway as described here may provide a novel therapeutic approach in the control of tumor progression and invasion.
The amygdala is instrumental to a set of brain processes that lead to cocaine consumption, including those that mediate reward and drug craving. This study examined the volumes of the amygdala and hippocampus in cocaine-addicted subjects and matched healthy controls and determined that the amygdala but not the hippocampus was significantly reduced in volume. The right-left amygdala asymmetry in control subjects was absent in the cocaine addicts. Topological analysis of amygdala isosurfaces (population averages) revealed that the isosurface of the cocaine-dependent group undercut the anterior and superior surfaces of the control group, implicating a difference in the corticomedial and basolateral nuclei. In cocaine addicts, amygdala volume did not correlate with any measure of cocaine use. The amygdala symmetry coefficient did correlate with baseline but not cocaine-primed craving. These findings argue for a condition that predisposes the individual to cocaine dependence by affecting the amygdala, or a primary event early in the course of cocaine use.
Parkinson's disease (PD; OMIM #168600) is the second most common neurodegenerative disorder in the Western world and presents as a progressive movement disorder. The hallmark pathological features of PD are loss of dopaminergic neurons from the substantia nigra and neuronal intracellular Lewy body inclusions. Parkinsonism is typically sporadic in nature; however, several rare familial forms are linked to genetic loci, and the identification of causal mutations has provided insight into the disease process. PARK8, identified in 2002 by Funayama and colleagues, appears to be a common cause of familial PD. We describe here the cloning of a novel gene that contains missense mutations segregating with PARK8-linked PD in five families from England and Spain. Because of the tremor observed in PD and because a number of the families are of Basque descent, we have named this protein dardarin, derived from the Basque word dardara, meaning tremor.
Galectin-4, a member of the galectin family, is expressed in the epithelium of the alimentary tract. It has two tandemly-repeated carbohydrate recognition domains (CRDs), and specifically binds to a SO3-3Gal1-3GalNAc pyranoside with high affinity (Ideo et al., Glycobiology 12, 199-208, 2002). In this study, we found that galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, such as SB1a, SM3, SM4s, SB2, SM2a, and GM1, but not to glycosphingolipids with 3-O-sialylated Gal, such as sLc4Cer, snLc4Cer, GM3, GM2, and GM4, using both an enzyme-linked immunosorbent assay and a surface plasmon resonance assay. A confocal immunocytochemical assay showed that galectin-4 was colocalized with SB1a, GM1, and carcinoembryonic antigen (CEA) in the patches on the cell surface of human colon adenocarcinoma CCK-81 and LS174T cells. This localization was distinct from caveolin/VIP21 localization. Furthermore, immobilized galectin-4 promoted adhesion of CCK-81 cells through the sulfated glycosphingolipid, SB1a. CEA also bound to galectin-4 with KD value of 2 x 10-8 M by SPR, and co-immunoprecipitated with galectin-4 in LS174T cell lysates. These findings suggest that SB1a and CEA in the patches on the cell surface of human colon adenocarcinoma cells could be biological important ligands for galectin-4.
SUMMARY D-Arabinans, composed of D-arabinofuranose (D-Araf), dominate the structure of mycobacterial cell walls, in two settings, as part of lipoarabinomannan (LAM) and arabinogalactan (AG), each with markedly different structures and functions. Little is known of the complexity of their biosynthesis. -D-Arabinofuranosyl-1-monophosphoryldecaprenol (C50-P-Araf) is the only known sugar donor, and EmbA, B, and C, products of the paralogous genes embA, B, and C, the sites of resistance to the anti-tuberculosis drug, ethambutol (EMB), are the only known implicated enzymes; EmbA and B apparently contribute to the synthesis of AG whereas EmbC is reserved for the synthesis of LAM. The Emb proteins show no over-all similarity to any known proteins, beyond Mycobacterium and related species. However, functional motifs, equivalent to a proline-rich motif of several bacterial Polysaccharide Co-Polymerases and a superfamily of glycosyltransferases (GT-C), were found. Site-directed mutagenesis in GT-C resulted in complete ablation of LAM synthesis. Point mutations in three amino acids of the proline motif of EmbC resulted in marked reduction of LAM-arabinan synthesis, and accumulation of an unknown intermediate and of the known precursor, lipomannan. Yet the pattern of the differently linked D-Araf units observed in wild type LAM-arabinan was largely retained in the proline motif mutants. The results allow for the presentation of a unique model of arabinan synthesis.
HIV/SIV envelope glycoproteins mediate the first steps in viral infection. They are trimers of a membrane-anchored polypeptide chain, cleaved into two fragments known as gp120 and gp41. The structure of HIV gp120 bound with receptor (CD4) has been known for some time. We have now determined the structure of a fully glycosylated SIV gp120 envelope glycoprotein in an unliganded conformation by X-ray crystallography at 4.0 Å resolution. We describe here our experimental and computational approaches, which may be relevant to other resolution-limited crystallographic problems. Key issues were attention to details of beam geometry mandated by small, weakly diffracting crystals, and choice of strategies for phase improvement, starting with two isomorphous derivatives and including multicrystal averaging. We validated the structure by analyzing composite omit maps, averaged among three distinct crystal lattices, and by calculating model-based, SeMet anomalous difference maps. There are at least four ordered sugars on many of the thirteen oligosaccharides
Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines.
Human embryonic stem cells (hESCs) self-renew indefinitely and give rise to derivatives of all three primary germ layers, yet little is known about the signaling cascades that govern their pluripotent character. Because it plays a prominent role in the early cell fate decisions of embryonic development, we have examined the role of TGF superfamily signaling in hESCs. We found that, in undifferentiated cells, the TGF/activin/nodal branch is activated (through the signal transducer SMAD2/3) while the BMP/GDF branch (SMAD1/5) is only active in isolated mitotic cells. Upon early differentiation, SMAD2/3 signaling is decreased while SMAD1/5 signaling is activated. We next tested the functional role of TGF/activin/nodal signaling in hESCs and found that it is required for the maintenance of markers of the undifferentiated state. We extend these findings to show that SMAD2/3 activation is required downstream of WNT signaling, which we have previously shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly, we show that in ex vivo mouse blastocyst cultures, SMAD2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a crucial role for TGF signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGF and WNT signaling in these contexts.
TSG-6 protein, upregulated in inflammatory lesions and in the ovary during ovulation, shows antiinflammatory activity and plays an essential role in female fertility. Studies in murine models of acute inflammation and experimental arthritis demonstrated that TSG-6 has a strong antiinflammatory and chondroprotective effect. TSG-6 protein is comprised of the N-terminal link module that binds hyaluronan, and a C-terminal CUB domain, present in a variety of proteins. Interactions between the isolated link module and hyaluronan have been studied extensively, but little is known about the binding of full-length TSG-6 protein to hyaluronan and other glycosaminoglycans. We show that TSG-6 protein and hyaluronan, in a temperature-dependent fashion, form a stable complex that is resistant to dissociating agents. The formation of such stable complexes may underlie the activities of TSG-6 protein in inflammation and fertility, e.g., the TSG-6-dependent cross-linking of hyaluronan in the cumulus cell-oocyte complex during ovulation. Because adhesion to hyaluronan is involved in cell trafficking in inflammatory processes, we also studied the effect of TSG-6 on cell adhesion. TSG-6 binding to immobilized hyaluronan did not interfere with subsequent adhesion of lymphoid cells. In addition to immobilized hyaluronan, full-length TSG-6 also binds free hyaluronan and all chondroitin sulfate isoforms under physiological conditions. These interactions may contribute to the localization of TSG-6 in cartilage and to its chondroprotective and antiinflammatory effects in models of arthritis.
Hox genes encode transcription factors that control spatial patterning during embryogenesis. To date, downstream targets of Hox genes have proven difficult to identify. Here, we describe studies designed to identify target genes under the control of the murine transcription factor Hoxc8. We used a mouse 16,463 gene oligonucleotide microarray to identify mRNAs whose expression was altered by the overexpression of Hoxc8 in C57BL/6J mouse embryo fibroblasts (MEF) in cell culture (in vitro). We identified a total of 34 genes whose expression was changed by 2-fold or greater: 16 genes were up-regulated, and 18 genes were down-regulated. The majority of genes encoded proteins involved in critical biological processes, such as cell adhesion, migration, metabolism, apoptosis, and tumorigenesis. Two genes showed high levels of regulation: (i) secreted phosphoprotein 1 (Spp1), also known as osteopontin (OPN), was down-regulated 4.8-fold, and (ii) frizzled homolog 2 (Drosophila) (Fzd2) was up-regulated 4.4-fold. Chromatin immunoprecipitation (ChIP) analysis confirmed the direct interaction between the OPN promoter and Hoxc8 protein in vivo, supporting the view that OPN is a direct transcriptional target of Hoxc8.
There is increasing evidence for intercellular trafficking of macromolecules through plasmodesmata (PD) during plant development. Here we study the ability of PD to traffic proteins during embryogenesis and early seedling development in Arabidopsis. Transgenic lines that induce GFP expression only in meristems, MSG (meristem-specific GFP), were used to monitor GFP movement. Cell-to-cell movement of different-sized GFP reporters reveals that embryos and young seedlings traffic proteins at least 54 kDa in size. Although 27-kDa soluble GFP (1xsGFP) freely moves between cells throughout the entire embryo during all stages analyzed, 2xsGFP movement becomes more restricted as development proceeds. After germination, cells near the apical meristem in seedlings show a higher size exclusion limit (SEL), whereas the SEL becomes more restricted as surrounding tissues develop identities. Although 1xsGFP moves throughout leaf primordia, as the leaf develops only the basal part of leaf petioles, main vascular tissues, and leaf veins (not blades) allow 1xsGFP movement. Although previous studies showed that embryos allow movement of small symplastic tracers (0.5 kDa), the present data demonstrate that the embryo constitutes a single symplast that allows transport of macromolecules as well. Even 2xsGFP moves from its site of expression at the apical meristem in embryos and seedlings, yet the extent of movement is more limited than 1xsGFP. Thus, PD have distinct SELs in different subregions of the embryo and seedling. These studies support the general concept that PD in younger tissues are more dilated and less restrictive than PD in older (nonvascular) tissues.
Mutations in the human parkin gene cause autosomal recessive juvenile parkinsonism, a heritable form of Parkinson's disease (PD). To determine whether mutations in the mouse parkin gene (Park2) also result in a parkinsonian phenotype, we generated mice with a targeted deletion of parkin exon 2. Using an extensive behavioral screen, we evaluated neurological function, motor ability, emotionality, learning, and memory in aged Parkin-deficient mice. The behavioral profile of Parkin-deficient mice on a B6;129S4 genetic background was strikingly similar to that of control mice, and most differences were not reproducible by using coisogenic mice on a 129S4 genetic background. Moreover, catecholamine levels in the striatum, olfactory bulb, and spinal cord of Parkin-deficient mice were normal. In contrast to previous studies using independently generated Parkin-deficient mice, we found no evidence for nigrostriatal, cognitive, or noradrenergic dysfunction. Understanding why Parkin-deficient mice do not exhibit robust signs of parkinsonism could advance knowledge and treatment of PD.
Synthetic prions were produced in our laboratory by using recombinant mouse prion protein (MoPrP) composed of residues 89-230. The first mouse synthetic prion strain (MoSP1) was inoculated into transgenic (Tg) 9949 mice expressing N-terminally truncated MoPrP(23-88) and WT FVB mice expressing full-length MoPrP. On first and second passage in Tg9949 mice, MoSP1 prions caused disease in 516 ± 27 and 258 ± 25 days, respectively; numerous, large vacuoles were found in the brainstem and gray matter of the cerebellum. MoSP1 prions passaged in Tg9949 mice were inoculated into FVB mice; on first and second passage, the FVB mice exhibited incubation times of 154 ± 4 and 130 ± 3 days, respectively. In FVB mice, vacuolation was less intense but more widely distributed, with numerous lesions in the hippocampus and cerebellar white matter. This constellation of widespread neuropatho-logic changes was similar to that found in FVB mice inoculated with Rocky Mountain Laboratory (RML) prions, a strain derived from a sheep with scrapie. Conformational stability studies showed that the half-maximal GdnHCl (Gdn1/2) concentration for denaturation of MoSP1 prions passaged in Tg9949 mice was 4.2 M; passage in FVB mice reduced the Gdn1/2 value to 1.7 M. RML prions passaged in either Tg9949 or FVB mice exhibited Gdn1/2 values of 1.8 M. The incubation times, neuropathological lesion profiles, and Gdn1/2 values indicate that MoSP1 prions differ from RML and many other prion strains derived from sheep with scrapie and cattle with bovine spongiform encephalopathy.
The -proteobacterium Shewanella oneidensis strain MR-1 is a metabolically versatile organism that can reduce a wide range of organic compounds, metal ions, and radionuclides. Similar to most other sequenced organisms, 40% of the predicted ORFs in the S. oneidensis genome were annotated as uncharacterized "hypothetical" genes. We implemented an integrative approach by using experimental and computational analyses to provide more detailed insight into gene function. Global expression profiles were determined for cells after UV irradiation and under aerobic and suboxic growth conditions. Transcriptomic and proteomic analyses confidently identified 538 hypothetical genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all predicted hypothetical proteins). Publicly available analysis tools and databases and the expression data were applied to improve the annotation of these genes. The annotation results were scored by using a seven-category schema that ranked both confidence and precision of the functional assignment. We were able to identify homologs for nearly all of these hypothetical proteins (97%), but could confidently assign exact biochemical functions for only 16 proteins (category 1; 3%). Altogether, computational and experimental evidence provided functional assignments or insights for 240 more genes (categories 2–5; 45%). These functional annotations advance our understanding of genes involved in vital cellular processes, including energy conversion, ion transport, secondary metabolism, and signal transduction. We propose that this integrative approach offers a valuable means to undertake the enormous challenge of characterizing the rapidly growing number of hypothetical proteins with each newly sequenced genome.
We use the pattern of intron conservation in 684 groups of orthologs from seven fully sequenced eukaryotic genomes to provide maximum likelihood estimates of the number of introns present in the same orthologs in various eukaryotic ancestors. We find: (i) intron density in the plant–animal ancestor was high, perhaps two-thirds that of humans and three times that of Drosophila; and (ii) intron density in the ancestral bilateran was also high, equaling that of humans and four times that of Drosophila. We further find that modern introns are generally very old, with two-thirds of modern bilateran introns dating to the ancestral bilateran and two-fifths of modern plant, animal, and fungus introns dating to the plant–animal ancestor. Intron losses outnumber gains over a large range of eukaryotic lineages. These results show that early eukaryotic gene structures were very complex, and that simplification, not embellishment, has dominated subsequent evolution.
The high prevalence of Duffy negativity (lack of the Duffy blood group antigen) among human populations in sub-Saharan Africa has been used to argue that Plasmodium vivax originated on that continent. Here, we investigate the phylogenetic relationships among 10 species of Plasmodium that infect primates by using three genes, two nuclear (-tubulin and cell division cycle 2) and a gene from the plastid genome (the elongation factor Tu). We find compelling evidence that P. vivax is derived from a species that inhabited macaques in Southeast Asia. Specifically, those phylogenies that include P. vivax as an ancient lineage from which all of the macaque parasites could originate are significantly less likely to explain the data. We estimate the time to the most recent common ancestor at four neutral gene loci from Asian and South American isolates (a minimum sample of seven isolates per locus). Our analysis estimates that the extant populations of P. vivax originated between 45,680 and 81,607 years ago. The phylogeny and the estimated time frame for the origination of current P. vivax populations are consistent with an "out of Asia" origin for P. vivax as hominoid parasite. The current debate regarding how the Duffy negative trait became fixed in Africa needs to be revisited, taking into account not only human genetic data but also the genetic diversity observed in the extant P. vivax populations and the phylogeny of the genus Plasmodium.
To elucidate cellular machinery on a global scale, we performed a multiple comparison of the recently available protein–protein interaction networks of Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae. This comparison integrated protein interaction and sequence information to reveal 71 network regions that were conserved across all three species and many exclusive to the metazoans. We used this conservation, and found statistically significant support for 4,645 previously undescribed protein functions and 2,609 previously undescribed protein interactions. We tested 60 interaction predictions for yeast by two-hybrid analysis, confirming approximately half of these. Significantly, many of the predicted functions and interactions would not have been identified from sequence similarity alone, demonstrating that network comparisons provide essential biological information beyond what is gleaned from the genome.
The transplantation of somatic cell nuclei to enucleated eggs has shown that genes can be reprogrammed to an embryonic pattern of expression, thereby indicating a reversal of their epigenetic state. However, in Xenopus nuclear transfer experiments using both endoderm and neuroectoderm donor cells, we have observed substantial overexpression of donor cell type-specific genes, both spatially and temporally, in the wrong cell type in some nuclear transplant embryos. For example, more than half of the embryos prepared from transplanted neuroectoderm nuclei overexpressed the neuroectodermal marker gene Sox2 to an excessive level in their endoderm cells. Because, in Xenopus, there is no transcription for the first 12 cell cycles, some somatic cell nuclei must remember a developmentally activated gene state and transmit this to their mitotic progeny in the absence of the conditions that induced that state. We also find that donor cell-specific genes are transcribed at an earlier stage than normal in an inappropriate cell type. This phenomenon of epigenetic memory applies to genes that are transcribed in donor nuclei; it does not influence those genes that are competent to be transcribed in nuclear transplant embryo tissue, but were not actually transcribed in donor nuclei at the time of nuclear transfer. We conclude that an epigenetic memory is established in differentiating somatic cells and applies to genes that are in a transcriptionally active state.
Abstract
Two papers in recent issue of Developmental Cell (Glise et al. 2005 and Gorfinkiel et al. 2005) have shown that Shifted, a Drosophila ortholog of Wnt Inhibitory Factor (WIF), modulates the distribution of Hedgehog protein in the wing imaginal disc through a Wnt-independent mechanism.
Experimental studies in insects have shown how sperm competition can be a potent selective force acting on an array of male reproductive traits [1, 2, 3 and 4]. However, the role of sperm quality in determining paternity in insects has been neglected, despite the fact that sperm quality has been shown to influence the outcome of sperm competition in vertebrates [5, 6, 7 and 8]. A recent comparative analysis found that males of polyandrous insect species show a higher proportion of live sperm in their stores [9]. Here, we test the hypothesis that sperm viability influences paternity at the within-species level. We use the cricket Teleogryllus oceanicus to conduct sperm competition trials involving prescreened males that differ in the viability of their sperm. We find that paternity success is determined by the proportion of live sperm in a male's ejaculate. Furthermore, we were able to predict the paternity patterns observed on the basis of the males' relative representation of viable sperm in the female's sperm-storage organ. Our findings provide the first experimental evidence for the theory that sperm competition selects for higher sperm quality in insects. Between-male variation in sperm quality needs to be considered in theoretical and experimental studies of insect sperm competition.
Abstract
Divergent reproductive interests of males and females often cause sexual conflict [1 and 2]. Males of many species manipulate females by transferring seminal fluids that boost female short-term fecundity while decreasing their life expectancy and future reproductivity [3 and 4]. The life history of ants, however, is expected to reduce sexual conflict; whereas most insect females show repeated phases of mating and reproduction, ant queens mate only during a short period early in life and undergo a lifelong commitment to their mates by storing sperm [5]. Furthermore, sexual offspring can only be reared after a sterile worker force has been built up [5]. Therefore, the males should also profit from a long female lifespan. In the ant Cardiocondyla obscurior, mating indeed has a positive effect on the lifetime reproductive success of queens. Queens that mated to either one fertile or one sterilized male lived considerably longer and started laying eggs earlier than virgin queens. Only queens that received viable sperm from fertile males showed increased fecundity. The lack of a trade-off between fecundity and longevity is unexpected, given evolutionary theories of aging [6]. Our data instead reveal the existence of sexual cooperation in ants.
Abstract
Background: In many female insects, peptides transferred in the seminal fluid induce postmating responses (PMR), such as a drastic increase of egg laying and reduction of receptivity (readiness to mate). In Drosophila melanogaster, sex-peptide (SP) elicits short- and long-term PMR, but only the latter in the presence of stored sperm (sperm effect).
Results: Here, we elucidate the interaction between SP and sperm by immunofluorescence microscopy. Transgenic males were used to study the effects of SP modification on the PMR of females in vivo. We report that SP binds to sperm with its N-terminal end. In females, the C-terminal part of SP known to be essential to induce the PMR is gradually released from stored sperm by cleavage at a trypsin cleavage site, thus prolonging the PMR. These findings are confirmed by analyzing the PMR elicited by males containing transgenes encoding modified SPs. SP lacking the N-terminal end cannot bind, and SP without the trypsin cleavage site binds permanently to sperm.
Conclusion: By binding to sperm tails, SP prolongs the PMR. Thus, besides a carrier for genetic information, sperm is also the carrier for SP. Binding to sperm may protect the peptide from degradation by proteases in the hemolymph and, thus, prolong its half-life. Longer sperm tails may transfer more SP and thus increase the reproductive fitness of the male. We suggest that this could explain the excessive length of sperm tails in some Drosophila species.
Hair graying is the most obvious sign of aging in humans, yet its mechanism is largely unknown. Here, we used melanocyte-tagged transgenic mice and aging human hair follicles to demonstrate that hair graying is caused by defective self-maintenance of melanocyte stem cells. This process is accelerated dramatically with Bcl2 deficiency, which causes selective apoptosis of melanocyte stem cells, but not of differentiated melanocytes, within the niche at their entry into the dormant state. Furthermore, physiologic aging of melanocyte stem cells was associated with ectopic pigmentation or differentiation within the niche, a process accelerated by mutation of the melanocyte master transcriptional regulator Mitf.
Is collagen just the workhorse of the extracellular matrix with few roles other than merely giving the body structural platforms? Hardly so. The collagen family has now swelled to at least 27 distinct members and thus is full of promise for functional diversity (1). Granted, some collagens belong to the best known and most abundant structural proteins. However, collagens also control sophisticated organ or tissue functions by means which are unexpected, fascinating, and often bewildering. Like other matrix macromolecules, collagens are functional only after aggregating into tissue suprastructures. They form complex alloys or composites containing not only several collagen types but also other kinds of molecules. Even tiny fractions of "minor" collagens astoundingly control suprastructural assembly and thus cell-matrix interactions dictating cellular activities such as growth, survival, differentiation, gene expression, and metabolism.
This minireview will focus on a growing subgroup of collagens, the collagenous transmembrane proteins, which have dual functions as cell surface receptors or as matrix components. If their extracellular domains are cast off by limited proteolysis, they can begin to signal to cells as soluble molecules. Collagenous transmembrane proteins are widely expressed and are involved in cell adhesion, epithelial-mesenchymal interactions during morphogenesis, neuromuscular signaling, and host defense against microbial agents. Correspondingly, they are associated with genetic and acquired human diseases, e.g. epidermolysis bullosa, ectodermal dysplasias, bullous pemphigoid, or Alzheimer disease, and mouse models implicate them in the etiology of further pathological conditions.
Small interfering RNA (siRNA) is normally designed to silence preselected known genes. Such selections are inevitably prone to bias as a result of limited knowledge about the biological process, transcript identity, and functions. A library that contains all permutations of siRNA could avoid such problems. In this paper, it is shown that 5 x 107 siRNA-encoding plasmids can be constructed in a single tube by using vectors with two mutated RNA polymerase III promoters arranged in a convergent manner. Such a library was used to carry out genomewide screening of functional genes in a phenotype-driven manner. Multiple siRNAs that induce a significant increase of cell proliferation speed were identified.
