Optical control of sphingolipid biosynthesis using photoswitchable sphingosines.

Sphingolipid metabolism comprises a complex interconnected web of enzymes, metabolites, and modes of regulation that influence a wide range of cellular and physiological processes. Deciphering the biological relevance of this network is challenging as numerous intermediates of sphingolipid metabolism are short-lived molecules with often opposing biological activities. Here, we introduce clickable, azobenzene-containing sphingosines, termed caSphs, as light-sensitive substrates for sphingolipid biosynthesis.

Optical control of sphingolipid biosynthesis using photoswitchable sphingosines.

Sphingolipid metabolism comprises a complex interconnected web of enzymes, metabolites and modes of regulation that influence a wide range of cellular and physiological processes. Deciphering the biological relevance of this network is challenging as numerous intermediates of sphingolipid metabolism are short-lived molecules with often opposing biological activities. Here, we introduce clickable, azobenzene-containing sphingosines, termed s, as light-sensitive substrates for sphingolipid biosynthesis.

Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co-Condensation of Wnt Signalodroplets.

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of ≈300 nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clamp fusions.

Time dependent differential regulation of a novel long non-coding natural antisense RNA during long-term memory formation.

Long natural antisense transcripts (NATs) have been demonstrated in significant numbers in a variety of eukaryotic organisms. They are particularly prevalent in the nervous system suggesting their importance in neural functions. However, the precise physiological roles of the overwhelming majority of long NATs remain unclear.

In cell Gd-based site-directed spin labeling and EPR spectroscopy of eGFP.

Label-based functional studies of biomolecules in their native environment require labeling reactions inside living cells. In cell spin labeling using alkyne-azide click chemistry with a Gd3+-DOTAM-azide complex is shown to provide high spin label stability and narrow EPR lines for EPR spectroscopic detection of a spin labeled protein in living cells at ambient temperatures.

Ceramides bind VDAC2 to trigger mitochondrial apoptosis.

Ceramides draw wide attention as tumor suppressor lipids that act directly on mitochondria to trigger apoptotic cell death. However, molecular details of the underlying mechanism are largely unknown. Using a photoactivatable ceramide probe, we here identify the voltage-dependent anion channels VDAC1 and VDAC2 as mitochondrial ceramide binding proteins.

Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides.

Ceramides are central intermediates of sphingolipid metabolism that also function as potent messengers in stress signaling and apoptosis. Progress in understanding how ceramides execute their biological roles is hampered by a lack of methods to manipulate their cellular levels and metabolic fate with appropriate spatiotemporal precision. Here, we report on clickable, azobenzene-containing ceramides, caCers, as photoswitchable metabolic substrates to exert optical control over sphingolipid production in cells.

A CREB2-targeting microRNA is required for long-term memory after single-trial learning.

Although single-trial induced long-term memories (LTM) have been of major interest in neuroscience, how LTM can form after a single episode of learning remains largely unknown. We hypothesized that the removal of molecular inhibitory constraints by microRNAs (miRNAs) plays an important role in this process. To test this hypothesis, first we constructed small non-coding RNA (sncRNA) cDNA libraries from the CNS of Lymnaea stagnalis subjected to a single conditioning trial.

A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7.

Ceramides are central intermediates of sphingolipid metabolism with dual roles as mediators of cellular stress signaling and mitochondrial apoptosis. How ceramides exert their cytotoxic effects is unclear and their poor solubility in water hampers a search for specific protein interaction partners. Here, we report the application of a photoactivatable and clickable ceramide analog, pacCer, to identify ceramide binding proteins and unravel the structural basis by which these proteins recognize ceramide.

The temporal expression profile of a Nos3-related natural antisense RNA in the brain suggests a possible role in neurogenesis.

Experimental work over the past several years has revealed an unexpected abundance of long natural antisense transcripts (NATs) in eukaryotic species. In light of the proposed role of such RNA molecules in the regulation of gene expression in the brain, attention is now focused on specific examples of neuronal NATs. Of particular interest are NATs that are complementary to mRNAs encoding nitric oxide synthase (NOS), the enzyme responsible for production of the important gaseous neurotransmitter nitric oxide (NO).

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases.

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear.

ER residency of the ceramide phosphoethanolamine synthase SMSr relies on homotypic oligomerization mediated by its SAM domain.

SMSr/SAMD8 is an ER-resident ceramide phosphoethanolamine synthase with a critical role in controlling ER ceramides and suppressing ceramide-induced apoptosis in cultured cells. SMSr-mediated ceramide homeostasis relies on the enzyme's catalytic activity as well as on its N-terminal sterile α-motif or SAM domain. Here we report that SMSr-SAM is structurally and functionally related to the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane.

Orthogonal spin labeling using click chemistry for in vitro and in vivo applications.

Site-directed spin labeling for EPR- and NMR spectroscopy has mainly been achieved exploiting the specific reactivity of cysteines. For proteins with native cysteines or for in vivo applications, an alternative coupling strategy is required. In these cases click chemistry offers major benefits by providing a fast and highly selective, biocompatible reaction between azide and alkyne groups.

Diverting CERT-mediated ceramide transport to mitochondria triggers Bax-dependent apoptosis.

A deregulation of ceramide biosynthesis in the endoplasmic reticulum (ER) is frequently linked to induction of mitochondrial apoptosis. Although in vitro studies suggest that ceramides might initiate cell death by acting directly on mitochondria, their actual contribution to the apoptotic response in living cells is unclear. Here, we have analyzed the consequences of targeting the biosynthetic flow of ceramides to mitochondria using a ceramide transfer protein (encoded by COL4A3BP) equipped with an OMM anchor, mitoCERT.

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases.

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear.

A novel long non-coding natural antisense RNA is a negative regulator of Nos1 gene expression.

Long non-coding natural antisense transcripts (NATs) are widespread in eukaryotic species. Although recent studies indicate that long NATs are engaged in the regulation of gene expression, the precise functional roles of the vast majority of them are unknown. Here we report that a long NAT (Mm-antiNos1 RNA) complementary to mRNA encoding the neuronal isoform of nitric oxide synthase (Nos1) is expressed in the mouse brain and is transcribed from the non-template strand of the Nos1 locus.

Cu(II)-catalyzed domino reaction of 2H-azirines with diazotetramic and diazotetronic acids. Synthesis of 2-substituted 2H-1,2,3-triazoles.

The Cu(II)-catalyzed addition of two molecules of a 3-aryl-2H-azirine to diazotetramic or diazotetronic acids proceeds as a domino reaction with the formation of 1,2,3-triazole derivatives with ortho-fused (pyrrolo[3,4-b]pyrrol or furo[3,4-b]pyrrol) and spiro-cyclic (1-oxa-4,7-diazaspiro[4.4]nonane or 1,7-dioxa-4-azaspiro[4.4]nonane) substituents at the N2 position.

Axonal trafficking of an antisense RNA transcribed from a pseudogene is regulated by classical conditioning.

Natural antisense transcripts (NATs) are endogenous RNA molecules that are complementary to known RNA transcripts. The functional significance of NATs is poorly understood, but their prevalence in the CNS suggests a role in brain function. Here we investigated a long NAT (antiNOS-2 RNA) associated with the regulation of nitric oxide (NO) production in the CNS of Lymnaea, an established model for molecular analysis of learning and memory.

Specific DNA duplex formation at an artificial lipid bilayer: towards a new DNA biosensor technology.

A novel technique is described which comprises a base-specific DNA duplex formation at a lipid bilayer-H(2) O-phase boundary layer. Two different probes of oligonucleotides both carrying a double-tailed lipid at the 5'-terminus were incorporated into stable artificial lipid bilayers separating two compartments (cis/trans-channel) of an optically transparent microfluidic sample carrier with perfusion capabilities. Both the cis- and trans-channels are filled with saline buffer.

Atypical guanylyl cyclase from the pond snail Lymnaea stagnalis: cloning, sequence analysis and characterization of expression.

Soluble guanylyl cyclases (sGCs) are traditionally recognized as the main molecular receptor for nitric oxide (NO), a gaseous transmitter involved in many functions of the nervous system. Some sGCs are however insensitive to NO and therefore are known as atypical. Although atypical sGCs have been shown to exist in both vertebrate and invertebrate nervous systems, our understanding of their functional role is incomplete.

Acetylcholine binding protein of mollusks is unlikely to act as a regulator of cholinergic neurotransmission at neurite-neurite synaptic sites in vivo.

A population of glial cells in the central nervous system of the gastropod mollusk Lymnaea stagnalis produces a soluble protein that specifically binds acetylcholine. This protein is named the acetylcholine binding protein (AChBP). Experiments performed in vitro indicated that AChBP inactivates released acetylcholine at cholinergic synapses.

Hyperphagia and increased meal size are responsible for weight gain in rats treated sub-chronically with olanzapine.

Rationale: Atypical antipsychotic-induced weight gain is a significant impediment in the treatment of schizophrenia.

Objectives: In a putative model of antipsychotic drug-induced weight gain, we investigated the effects of sub-chronic olanzapine on body weight, meal patterns, the expression of genes encoding for hypothalamic feeding-related neuropeptides and the contribution of hyperphagia to olanzapine-induced weight gain in rats.

Materials And Methods: In experiment 1, female rats received either olanzapine (1 mg/kg, p.

Novel noncoding antisense RNA transcribed from human anti-NOS2A locus is differentially regulated during neuronal differentiation of embryonic stem cells.

Here, we report on the discovery of a locus in the human genome, which evolved by gene duplication followed by an internal DNA inversion. This locus exhibits high sequence similarity to the gene for the inducible isoform of NOS protein (NOS2A) and is transcribed into a noncoding RNA containing a region of significant antisense homology with the NOS2A mRNA. We show that this antisense transcript (anti-NOS2A RNA) is expressed in different types of brain tumors, including meningiomas and glioblastomas.

Characterization of NO-sensitive guanylyl cyclase: expression in an identified interneuron involved in NO-cGMP-dependent memory formation.

In a number of neuronal models of learning signalling by endogenous nitric oxide (NO), produced by the enzyme NO synthase (NOS), is essential for the formation of long-term memory (LTM). For example, in the molluscan model system Lymnaea, NO is required for LTM formation in the first few hours after one-trial reward conditioning. Furthermore, conditioning leads to transient up-regulation of the NOS gene in identified modulatory neurons, the cerebral giant cells (CGCs), which are known to be involved in LTM formation.

Natural antisense RNAs in the nervous system.

Natural antisense RNAs are endogenous molecules that are complementary to RNA transcripts of already established function. They were discovered first in prokaryotes in which they are now recognised as an important component of molecular mechanisms involved in the regulation of gene expression. Recently, through the cumulative efforts of molecular biologists and bioinformaticians, natural antisense RNAs have been demonstrated in significant numbers in eukaryotic systems also.