Morpholino oligos: making sense of antisense?

Since morpholino oligos were first introduced as a means to inhibit gene function in embryos, in the Spring of 2000, they have been tested in a range of model organisms, including sea urchin, ascidian, zebrafish, frog, chick, and mouse. This review surveys the results of these studies and examines the successes and limitations of the approach for targeting maternal and zygotic gene function. The evidence so far suggests that, with careful controls, morpholinos provide a relatively simple and rapid method to study gene function.     

The regulation of autophagy by calcium signals: Do we have a consensus?

Macroautophagy (hereafter called ‘autophagy’) is a cellular process for degrading and recycling cellular constituents, and for maintenance of cell function. Autophagy initiates via vesicular engulfment of cellular materials and culminates in their degradation via lysosomal hydrolases, with the whole process often being termed ‘autophagic flux’. Autophagy is a multi-step pathway requiring the interplay of numerous scaffolding and signalling molecules. In particular, orthologs of the family of ∼30 autophagy-regulating (Atg) proteins that were first characterised in yeast play essential roles in the initiation and processing of autophagic vesicles in mammalian cells. The serine/threonine kinase mTOR (mechanistic target of rapamycin) is a master regulator of the canonical autophagic response of cells to nutrient starvation. In addition, AMP-activated protein kinase (AMPK), which is a key sensor of cellular energy status, can trigger autophagy by inhibiting mTOR, or by phosphorylating other downstream targets. Calcium (Ca²⁺) has been implicated in autophagic signalling pathways encompassing both mTOR and AMPK, as well as in autophagy seemingly not involving these kinases. Numerous studies have shown that cytosolic Ca²⁺ signals can trigger autophagy. Moreover, introduction of an exogenous chelator to prevent cytosolic Ca²⁺ signals inhibits autophagy in response to many different stimuli, with suggestions that buffering Ca²⁺ affects not only the triggering of autophagy, but also proximal and distal steps during autophagic flux. Observations such as these indicate that Ca²⁺ plays an essential role as a pro-autophagic signal. However, cellular Ca²⁺ signals can exert anti-autophagic actions too. For example, Ca²⁺ channel blockers induce autophagy due to the loss of autophagy-suppressing Ca²⁺ signals. In addition, the sequestration of Ca²⁺ by mitochondria during physiological signalling appears necessary to maintain cellular bio-energetics, thereby suppressing AMPK-dependent autophagy. This article attempts to provide an integrated overview of the evidence for the proposed roles of various Ca²⁺ signals, Ca²⁺ channels and Ca²⁺ sources in controlling autophagic flux.     

Assessing the preferred solution conformation of an interacting sense–antisense (complementary) peptide pair

Sense peptides and corresponding antisense peptides, are capable of making specific interactions. Such interactions may result from inter-peptide side-chain/side-chain contacts or because peptides adopt mutually complementary three-dimensional shapes. Using a combined ¹H NMR spectroscopy/molecular modeling approach to study the interactions between one sense peptide and its corresponding antisense peptide, data are produced that provide clear support for the former hypothesis.     

Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR

Nucleic acid-based gene interference technologies represent promising strategies for specific inhibition of mRNA sequences of choice. Recently, small interfering RNAs have been implicated in inducing endogenous RNase of the RNA-induced silencing complex in the RNA interference pathway to inhibit gene expression and growth of several human viruses. We report down regulation of gene expression of E. coli gyrase A, an essential gene for DNA supercoiling and antibiotic susceptibility in BL21 (DE3) strain of E. coli, using Ribonuclease P based external guide sequence (EGS) technique. EGS directed against gyrase A gene that was cloned into pUC vector, which contains the ampicillin (Amp) resistance gene. The recombinant plasmid pT7EGyrA was transformed into BL21 (DE3) and inductions were performed using IPTG. RT-PCR experiment was done to investigate the down regulation of gyrase A gene. RT-PCR results demonstrated a significant decrease of gyrase A gene after 18 h of induction of the transformants. These experiments showed that the down regulation of the gene was seen after 18 h of induction than earlier hours of induction with IPTG suggesting inhibition of gyrase A gene with profound effect on cell viability. These results demonstrate the utility of EGS RNAs in gene therapy applications, by inhibiting the expression of essential proteins.     

Differential regulation of the calpain–calpastatin complex by the L-domain of calpastatin

Here we demonstrate that the presence of the L-domain in calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain–calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca^2 +] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which calpastatin levels are increased. Our findings show that the L-domain of calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.     

Modulation of human interferon-γ biosynthesis by antisense oligodeoxynucleotides

We investigated the inhibition of human interferon-γ (HuIFN-γ) production in cultures of lymphocytes with the use of the antisense strategy. Out of a series of antisense oligodeoxynucleotides (ODN) complementary to different regions of the HuIFN-γ gene, a 16-mer specific for a sequence including the translation initiation codon was the most effective. Here we describe a detailed protocol for the isolation of lymphocytes from buffy coats, the rational design of antisense ODN, and the monitoring of HuIFN-γ production of the antisense ODN-treated cells.     

Rank–rank hypergeometric overlap: identification of statistically significant overlap between gene-expression signatures

Comparing independent high-throughput gene-expression experiments can generate hypotheses about which gene-expression programs are shared between particular biological processes. Current techniques to compare expression profiles typically involve choosing a fixed differential expression threshold to summarize results, potentially reducing sensitivity to small but concordant changes. We present a threshold-free algorithm called Rank–rank Hypergeometric Overlap (RRHO). This algorithm steps through two gene lists ranked by the degree of differential expression observed in two profiling experiments, successively measuring the statistical significance of the number of overlapping genes. The output is a graphical map that shows the strength, pattern and bounds of correlation between two expression profiles. To demonstrate RRHO sensitivity and dynamic range, we identified shared expression networks in cancer microarray profiles driving tumor progression, stem cell properties and response to targeted kinase inhibition. We demonstrate how RRHO can be used to determine which model system or drug treatment best reflects a particular biological or disease response. The threshold-free and graphical aspects of RRHO complement other rank-based approaches such as Gene Set Enrichment Analysis (GSEA), for which RRHO is a 2D analog. Rank–rank overlap analysis is a sensitive, robust and web-accessible method for detecting and visualizing overlap trends between two complete, continuous gene-expression profiles. A web-based implementation of RRHO can be accessed at http://systems.crump.ucla.edu/rankrank/.     

The architecture of a mixed fungal–bacterial biofilm is modulated by quorum-sensing signals

Interkingdom communication is of particular relevance in polymicrobial biofilms. In this work, the ability of the fungus Ophiostoma piceae to form biofilms individually and in consortium with the bacterium Pseudomonas putida, as well as the effect of fungal and bacterial signal molecules on the architecture of the biofilms was evaluated. Pseudomonas putida KT2440 is able to form biofilms through the secretion of exopolysaccharides and two large extracellular adhesion proteins, LapA and LapF. It has two intercellular signalling systems, one mediated by dodecanoic acid and an orphan LuxR receptor that could participate in the response to AHL-type quorum sensing molecules (QSMs). Furthermore, the dimorphic fungus O. piceae uses farnesol as QSM to control its yeast to hyphae morphological transition. Results show for the first time the ability of this fungus to form biofilms alone and in mixed cultures with the bacterium. Biofilms were induced by bacterial and fungal QSMs. The essential role of LapA-LapF proteins in the architecture of biofilms was corroborated, LapA was induced by farnesol and dodecanol, while LapF by 3-oxo-C6-HSL and 3-oxo-C12-HSL. Our results indicate that fungal signals can induce a transient rise in the levels of the secondary messenger c-di-GMP, which control biofilm formation and architecture.     

Recognition of nuclear targeting signals by Karyopherin-β proteins

The Karyopherin-β family of nuclear transport factors mediates the majority of nucleocytoplasmic transport. Although each of the 19 Karyopherin-βs transports unique sets of cargos, only three classes of nuclear localization and export signals, or NLSs and NESs, have been characterized. The short basic classical-NLS was first discovered in the 1980s and their karyopherin-bound structures were first reported more than 10 years ago. More recently, structural and biophysical studies of Karyopherin-β2-cargo complexes led to definition of the complex and diverse PY-NLS. Structural knowledge of the leucine-rich NES is finally available more than 10 years after the discovery of its recognition by the exportin CRM1. We review recent findings relating to how these three classes of nuclear targeting signals are recognized by their Karyopherin-β nuclear transport factors.     

Dynamic regulation of lipid–protein interactions

We review the importance of helix motions for the function of several important categories of membrane proteins and for the properties of several model molecular systems. For voltage-gated potassium or sodium channels, sliding, tilting and/or rotational movements of the S4 helix accompanied by a swapping of cognate side-chain ion-pair interactions regulate the channel gating. In the seven-helix G protein-coupled receptors, exemplified by the rhodopsins, collective helix motions serve to activate the functional signaling. Peptides which initially associate with lipid-bilayer membrane surfaces may undergo dynamic transitions from surface-bound to tilted-transmembrane orientations, sometimes accompanied by changes in the molecularity, formation of a pore or, more generally, the activation of biological function. For single-span membrane proteins, such as the tyrosine kinases, an interplay between juxtamembrane and transmembrane domains is likely to be crucial for the regulation of dimer assembly that in turn is associated with the functional responses to external signals. Additionally, we note that experiments with designed single-span transmembrane helices offer fundamental insights into the molecular features that govern protein–lipid interactions.This article is part of a Special Issue entitled: Lipid–protein interactions.     

Metabolic regulation of citrate and iron by aconitases: role of iron–sulfur cluster biogenesis

Iron and citrate are essential for the metabolism of most organisms, and regulation of iron and citrate biology at both the cellular and systemic levels is critical for normal physiology and survival. Mitochondrial and cytosolic aconitases catalyze the interconversion of citrate and isocitrate, and aconitase activities are affected by iron levels, oxidative stress and by the status of the Fe–S cluster biogenesis apparatus. Assembly and disassembly of Fe–S clusters is a key process not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron sensing and RNA binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). This review discusses the central role of aconitases in intermediary metabolism and explores how iron homeostasis and Fe–S cluster biogenesis regulate the Fe–S cluster switch and modulate intracellular citrate flux.     

Nutriepigenetic regulation by folate–homocysteine–methionine axis: a review

Although normally folic acid is given during pregnancy, presumably to prevent neural tube defects, the mechanisms of this protection are unknown. More importantly it is unclear whether folic acid has other function during development. It is known that folic acid re-methylates homocysteine (Hcy) to methionine by methylene tetrahydrofolate reductase-dependent pathways. Folic acid also generates high-energy phosphates, behaves as an antioxidant and improves nitric oxide (NO) production by endothelial NO synthase. Interestingly, during epigenetic modification, methylation of DNA/RNA generate homocysteine unequivocally. The enhanced overexpression of methyl transferase lead to increased yield of Hcy. The accumulation of Hcy causes vascular dysfunction, reduces perfusion in the muscles thereby causing musculopathy. Another interesting fact is that children with severe hyperhomocysteinaemia (HHcy) have skeletal deformities, and do not live past teenage. HHcy is also associated with the progeria syndrome. Epilepsy is primarily caused by inhibition of gamma-amino-butyric-acid (GABA) receptor, an inhibitory neurotransmitter in the neuronal synapse. Folate deficiency leads to HHcy which then competes with GABA for binding on the GABA receptors. With so many genetic and clinical manifestations associated with folate deficiency, we propose that folate deficiency induces epigenetic alterations in the genes and thereby results in disease.     

Synthesis of antisense oligonucleotides containing 2′-O-psoralenylmethoxyalkyl adenosine for photodynamic regulation of point mutations in RNA

2′-O-Psoralen-conjugated antisense oligonucleotide was able to recognize a point mutation of mRNA. It had outstanding ability to photo-cross-link only to oligoribonucleotides (ORN) having a point mutation. This type of antisense molecule is the only one of its kind so far. To give high photo-cross-linking efficiency and sequence selectivity to antisense molecules, we synthesized novel photo-reactive oligonucleotides (2′-Ps-xom) containing psoralen at the 2′-O-position adenosine via an ethoxymethylene (2′-Ps-eom), propoxymethylene (2′-Ps-pom) and butoxymethylene (2′-Ps-bom) linker, respectively. We evaluated the photo-cross-linking efficiency and sequence selectivity in photo-cross-linking of 2′-Ps-xom to the complementary ORN and to an ORN having a mismatch base. Among them, 2′-Ps-eom exhibited superior photo-cross-linking efficiency with high sequence selectivity.     

Investigation of Gene Insertion–Deletion Polymorphism of the Gene for Angiotensin-Converting Enzyme in Populations of the Volga–Ural Region

Insertion–deletion polymorphism at the angiotensin I-converting enzyme (ACE) gene in populations of the Volga–Ural region was examined by means of polymerase chain reaction. The populations studied belong to the Finno-Ugric (Komis, Maris, Mordovians, and Udmurts), Turkic (Chuvashes, Tatars, and Bashkirs), and Eastern-Slavic (Russians) ethnic groups. Distribution patterns of allele and genotype frequencies of this polymorphic system in the examined region were characterized. Comparison of the obtained results with the literature data on the ACE gene polymorphism in other Caucasoid and Mongoloid populations revealed some trends in the ACE genotype frequency dynamics depending on the ethnicity of the populations.