Isothermal Titration Calorimetry Analysis of a Cooperative Riboswitch Using an Interdependent-Sites Binding Model.

Isothermal titration calorimetry (ITC) is a powerful biophysical tool to characterize energetic profiles of biomacromolecular interactions without any alteration of the underlying chemical structures. In this protocol, we describe procedures for performing, analyzing, and interpreting ITC data obtained from a cooperative riboswitch-ligand interaction.

Pre-mRNA splicing factor U2AF2 recognizes distinct conformations of nucleotide variants at the center of the pre-mRNA splice site signal.

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced UV-crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites, with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

A small RNA that cooperatively senses two stacked metabolites in one pocket for gene control.

Riboswitches are structured non-coding RNAs often located upstream of essential genes in bacterial messenger RNAs. Such RNAs regulate expression of downstream genes by recognizing a specific cellular effector. Although nearly 50 riboswitch classes are known, only a handful recognize multiple effectors. Here, we report the 2.60-Å resolution co-crystal structure of a class I type I preQ1-sensing riboswitch that reveals two effectors stacked atop one another in a single binding pocket. These effectors bind with positive cooperativity in vitro and both molecules are necessary for gene regulation in bacterial cells. Stacked effector recognition appears to be a hallmark of the largest subgroup of preQ1 riboswitches, including those from pathogens such as Neisseria gonorrhoeae. We postulate that binding to stacked effectors arose in the RNA World to closely position two substrates for RNA-mediated catalysis. These findings expand known effector recognition capabilities of riboswitches and have implications for antimicrobial development.

Arginine Forks Are a Widespread Motif to Recognize Phosphate Backbones and Guanine Nucleobases in the RNA Major Groove.

RNA recognition by proteins is central to biology. Here we demonstrate the existence of a recurrent structural motif, the "arginine fork", that codifies arginine readout of cognate backbone and guanine nucleobase interactions in a variety of protein-RNA complexes derived from viruses, metabolic enzymes, and ribosomes. Nearly 30 years ago, a theoretical arginine fork model was posited to account for the specificity between the HIV-1 Tat protein and TAR RNA. This model predicted that a single arginine should form four complementary contacts with nearby phosphates, yielding a two-pronged backbone readout. Recent high-resolution structures of TAR-protein complexes have unveiled new details, including (i) arginine interactions with the phosphate backbone and the major-groove edge of guanine and (ii) simultaneous cation-π contacts between the guanidinium group and flanking nucleobases. These findings prompted us to search for arginine forks within experimental protein-RNA structures retrieved from the Protein Data Bank. The results revealed four distinct classes of arginine forks that we have defined using a rigorous but flexible nomenclature. Examples are presented in the context of ribosomal and nonribosomal interfaces with analysis of arginine dihedral angles and structural (suite) classification of RNA targets. When arginine fork chemical recognition principles were applied to existing structures with unusual arginine-guanine recognition, we found that the arginine fork geometry was more consistent with the experimental data, suggesting the utility of fork classifications to improve structural models. Software to analyze arginine-RNA interactions has been made available to the community.

Analysis of a preQ1-I riboswitch in effector-free and bound states reveals a metabolite-programmed nucleobase-stacking spine that controls gene regulation.

Riboswitches are structured RNA motifs that recognize metabolites to alter the conformations of downstream sequences, leading to gene regulation. To investigate this molecular framework, we determined crystal structures of a preQ1-I riboswitch in effector-free and bound states at 2.00 Å and 2.65 Å-resolution. Both pseudoknots exhibited the elusive L2 loop, which displayed distinct conformations. Conversely, the Shine-Dalgarno sequence (SDS) in the S2 helix of each structure remained unbroken. The expectation that the effector-free state should expose the SDS prompted us to conduct solution experiments to delineate environmental changes to specific nucleobases in response to preQ1. We then used nudged elastic band computational methods to derive conformational-change pathways linking the crystallographically-determined effector-free and bound-state structures. Pathways featured: (i) unstacking and unpairing of L2 and S2 nucleobases without preQ1-exposing the SDS for translation and (ii) stacking and pairing L2 and S2 nucleobases with preQ1-sequestering the SDS. Our results reveal how preQ1 binding reorganizes L2 into a nucleobase-stacking spine that sequesters the SDS, linking effector recognition to biological function. The generality of stacking spines as conduits for effector-dependent, interdomain communication is discussed in light of their existence in adenine riboswitches, as well as the turnip yellow mosaic virus ribosome sensor.

Structure of HIV TAR in complex with a Lab-Evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.

Natural and lab-evolved proteins often recognize their RNA partners with exquisite affinity. Structural analysis of such complexes can offer valuable insight into sequence-selective recognition that can be exploited to alter biological function. Here, we describe the structure of a lab-evolved RNA recognition motif (RRM) bound to the HIV-1 trans-activation response (TAR) RNA element at 1.80 Å-resolution. The complex reveals a trio of arginines in an evolved ß2-ß3 loop penetrating deeply into the major groove to read conserved guanines while simultaneously forming cation-π and salt-bridge contacts. The observation that the evolved RRM engages TAR within a double-stranded stem is atypical compared to most RRMs. Mutagenesis, thermodynamic analysis and molecular dynamics validate the atypical binding mode and quantify molecular contributions that support the exceptionally tight binding of the TAR-protein complex (KD,App of 2.5 ± 0.1 nM). These findings led to the hypothesis that the ß2-ß3 loop can function as a standalone TAR-recognition module. Indeed, short constrained peptides comprising the ß2-ß3 loop still bind TAR (KD,App of 1.8 ± 0.5 µM) and significantly weaken TAR-dependent transcription. Our results provide a detailed understanding of TAR molecular recognition and reveal that a lab-evolved protein can be reduced to a minimal RNA-binding peptide.

Trafficking of excitatory amino acid transporter 2-laden vesicles in cultured astrocytes: a comparison between approximate and exact determination of trajectory angles.

A clear consensus concerning the mechanisms of intracellular secretory vesicle trafficking in astrocytes is lacking in the physiological literature. A good characterization of vesicle trafficking that may assist researchers in achieving that goal is the trajectory angle, defined as the angle between the trajectory of a vesicle and a line radial to the cell's nucleus. In this study, we provide a precise definition of the trajectory angle, describe and compare two methods for its calculation in terms of measureable trafficking parameters, and give recommendations for the appropriate use of each method. We investigated the trafficking of vesicles containing excitatory amino acid transporter 2 (EAAT2) fluorescently tagged with enhanced green fluorescent protein (EGFP) to quantify and validate the precision of each method. The motion of fluorescent puncta--taken to represent vesicles containing EAAT2-EGFP--was found to be typical of secretory vesicle trafficking. An exact method for calculating the trajectory angle of these puncta produced no error but required large computation time. An approximate method reduced the requisite computation time but produced an error depending on the inverse of the ratio of the punctum's initial distance from the nucleus centroid to its maximal displacement. Fitting this dependence to a power function allowed us to establish an exclusion distance from the centroid, beyond which the approximate method is less likely to produce an error above an acceptable 5%. We recommend that the exact method be used to calculate the trajectory angle for puncta closer to the nucleus centroid than this exclusion distance.

Method for the determination of trajectory angles of directional secretory vesicles in cultured astrocytes.

Astrocytes provide a principal pathway for glutamate uptake in the mammalian brain, a task accomplished by the powerful action of excitatory amino acid transporters (EAAT) 1 and 2. These transporters are synthesized within the endoplasmic reticulum and are then trafficked to the plasma membrane. The characteristics of their intracellular traffic within astrocytes have not been investigated. We monitored the trafficking of secretory vesicles laden with the recombinant fluorescent protein chimera of EAAT2 in cultured astrocytes. Such vesicles appeared as fluorescent puncta, and their trafficking parameters were obtained using original algorithms, which we describe here in detail. We determined the maximal displacement, average instantaneous speed, and trajectory angle of individual puncta/vesicles, with angles near 0° indicating radial movement directly away from or toward the nucleus and angles near 90° indicating tangential movement. Analysis of these trafficking parameters demonstrated that trafficking of EAAT2-laden vesicles has typical characteristics expected of the trafficking of secretory vesicles in cultured astrocytes. The distribution of trajectory angles for directional vesicles, i.e. those with a maximal displacement greater than 1 µm within the 40-s time-lapse imaging, was found to be unimodal, with angles near 0° being the most prominent (mode 7°). These measurements are in good agreement with previous measurements of trajectory angles of similar trafficking vesicles carrying cannabinoid receptor 1, evidencing the validity and robustness of our analytical approach and algorithms.

Submillimetre galaxies reside in dark matter haloes with masses greater than 3 × 10(11) solar masses.

The extragalactic background light at far-infrared wavelengths comes from optically faint, dusty, star-forming galaxies in the Universe with star formation rates of a few hundred solar masses per year. These faint, submillimetre galaxies are challenging to study individually because of the relatively poor spatial resolution of far-infrared telescopes. Instead, their average properties can be studied using statistics such as the angular power spectrum of the background intensity variations. A previous attempt at measuring this power spectrum resulted in the suggestion that the clustering amplitude is below the level computed with a simple ansatz based on a halo model. Here we report excess clustering over the linear prediction at arcminute angular scales in the power spectrum of brightness fluctuations at 250, 350 and 500 µm. From this excess, we find that submillimetre galaxies are located in dark matter haloes with a minimum mass, M(min), such that log(10)[M(min)/M(⊙)] = 11.5(+0.7)(-0.2) at 350 µm, where M(⊙) is the solar mass. This minimum dark matter halo mass corresponds to the most efficient mass scale for star formation in the Universe, and is lower than that predicted by semi-analytical models for galaxy formation.

Ganglion cell neurites in human idiopathic epiretinal membranes.

AIM: To identify and confirm the presence of neural elements in idiopathic epiretinal membranes removed from patients' eyes during vitrectomy with epiretinal membrane peeling. METHODS: Human epiretinal membranes from patients with no other known eye disease and of varying durations were labelled immunohistochemically with antibodies for neurofilament protein, laminin and either vimentin or GFAP; proteins expressed in ganglion cells, the inner limiting membrane (ILM), and Muller cells, respectively. RESULTS: Anti-neurofilament labelled neurites, presumed to originate from ganglion cells, were found in all 32 idiopathic epiretinal membranes examined. The neurites were only observed in regions of anti-vimentin or -GFAP labelled glial cells, both of which were observed embedded in anti-laminin labelled material assumed to originate from the ILM. CONCLUSIONS: We show that neurofilamentous processes, presumed to originate from retinal ganglion cells, are found universally in idiopathic epiretinal membranes, suggesting that the presence of these membranes is sufficient to stimulate neurite growth in the absence of trauma or disease. In addition, since neurites were invariably found in association with glial cells, the glia may play a permissive role in neurite growth both within the retina and into extra-retinal glial membranes.

A 5-HT(7) receptor-mediated depolarization in the anterodorsal thalamus. I. Pharmacological characterization.

Little is currently known regarding the electrophysiological response elicited by 5-hydroxytryptamine-7 (5-HT(7)) receptor stimulation in the brain. Previous anatomical studies have shown that the anterior thalamus expresses a high density of 5-HT7 receptors. Therefore, we used whole-cell recording techniques in the in vitro brain slices to examine the effects of serotonin on neurons of the anterodorsal nucleus of the thalamus (ADn). Bath application of 5-HT induces a large membrane depolarization and inward current in neurons of the ADn. Since these cells expressed 5-HT7 receptor mRNA, as determined by single-cell reverse transcriptase-polymerase chain reaction, we pharmacologically characterized the 5-HT receptor mediating this response. We found that the 5-HT1 and 5-HT7 agonists 5-carboxamidotryptamine (5-CT) and 5-methoxytryptamine mimicked the response to 5-HT, whereas the 5-HT2 agonist 2,5-dimethoxy-4-iodoamphetamine did not. Consistent with the involvement of a 5-HT7 receptor, 5-CT was approximately 18 times more potent than 5-HT. Furthermore, administration of the 5-HT(1A) and 5-HT7 agonist 8-hydroxydipropylaminotetralin mimicked and antagonized the effect of serotonin, suggesting it acted as a partial agonist. To determine if either the 5-HT1 or 5-HT7 receptor mediated the 5-HT-induced inward current, we used antagonists. We found that the 5-HT7 ligands ritanserin, methylsergide, LSD, and mesulergine could inhibit the 5-HT-induced inward current, whereas the 5-HT1 antagonist cyanopindolol had no effect. The pA(2) value determined for mesulergine closely approximated that expected for a 5-HT7 receptor. Finally, we found that bath application of the selective antagonist SB-269770 blocks the 5-HT-induced inward current. These results identify the receptor mediating the serotonin-induced membrane depolarization in the ADn as the 5-HT7 subtype.

A 5-HT(7) receptor-mediated depolarization in the anterodorsal thalamus. II. Involvement of the hyperpolarization-activated current I(h).

Previous studies have shown that 5-hydroxytryptamine (5-HT) can modulate the hyperpolarization-activated nonselective cation current (I(h)) to elicit a membrane depolarization in neurons. However, the receptor subtype involved in this response remains controversial. In the accompanying study, we have identified a 5-HT7 receptor-mediated depolarization in the anterodorsal nucleus of the thalamus (ADn). In the present study, we have examined the possible role of I(h) in mediating this 5-HT7 receptor-mediated depolarization. We used the blind tight-seal patch clamp technique to examine the ability of 5-HT to modulate I(h) in the ADn. We found that 5-HT induced a shift in the voltage dependence of I(h) to more depolarized potentials. The pharmacology of the receptor mediating this effect was consistent with that of a 5-HT7 receptor. Since the 5-HT7 receptor is coupled positively to adenylate cyclase, we examined the cAMP dependence of the 5-HT-induced modulation of I(h). Intracellular addition of cAMP mimicked and occluded the 5-HT response. Conversely, in the presence of the protein kinase inhibitors H-8 and staurosporine, ADn neurons still expressed a 5-HT-induced shift in the voltage dependence of I(h). These results suggest that 5-HT regulates I(h) in the ADn through a cAMP-dependent but protein kinase A (PKA)-independent mechanism. To determine the contribution of I(h) to the 5-HT7 receptor-mediated depolarization, we used the selective I(h) blocker ZD7288. This compound greatly reduced the depolarizing response elicited by activation of 5-HT7 receptors. We conclude that 5-HT7 receptors depolarize ADn neurons primarily by increasing I(h) through a cAMP-dependent, PKA-independent mechanism.

The state of the Innocenti Declaration targets in Italy.

If Italy is to meet the operational targets of the Innocenti Declaration, there will need to be changes in the way that breastfeeding is not only promoted but also protected and supported. During the celebration of World Breastfeeding Week in October 2000, a resolution was passed in the parliamentary Commissione per l'infanzia (Commission on Childhood) that committed the government to promoting, protecting, and supporting breastfeeding in a more active manner; to including breastfeeding as a health indicator; and to establishing national and regional goals for breastfeeding prevalence and duration. A next step that must be taken by the government is that of creating a national committee to coordinate the work. Progress toward accrediting a baby-friendly hospital needs to continue and must use the international standards as criteria. Recent action taken by the Ministry of Health and the court system is promising; however, full compliance of the International Code of Marketing of Breast-Milk Substitutes should be a goal for Italy and the rest of the European Union. The one area in which Italy seems to have fulfilled all of its obligations is the one concerning "imaginative legislation"; however, this shows that although legislative protection is important, it is not enough to ensure breastfeeding success.

Ribofuranosyl triazolone: a natural product herbicide with activity on adenylosuccinate synthetase following phosphorylation.

2,4-Dihydro-4-(beta-D-ribofuranosyl)-1,2,4(3H)-triazol-3-one (2) was identified as the principal phytotoxic component of a fermentation broth derived from an Actinomadura. The compound is a new natural product, but known by synthesis. Broad-spectrum herbicidal activity was demonstrated in greenhouse tests. Metabolite reversal studies suggested the target site was adenylosuccinate synthetase, which was confirmed by direct measurement of the activity of the 5'-phosphorylated derivative on the isolated enzyme.

New diffusion-edited NMR experiments to expedite the dereplication of known compounds from natural product mixtures.

[reaction: see text] We present two new diffusion-edited NMR experiments, improved DECODES and HETDECODES, that sort the constituents in a mixture by their individual diffusion coefficients. These experiments should allow the partial NMR spectral assignment and cursory structure elucidation of compounds in a complex mixture as an aid in the dereplication of known or nuisance compounds.

Calcium-independent afterdepolarization regulated by serotonin in anterior thalamus.

Previous studies have identified an afterdepolarization (ADP) in thalamocortical neurons that is mediated by an upregulation of the hyperpolarization-activated current I(h). This ADP has been suggested to play a key role in the generation of spindle oscillations. In the lateral geniculate nucleus, upregulation of I(h) has been shown to be signaled by a rise in intracellular calcium leading to the activation of adenylate cyclase and formation of cAMP. However, it is unclear how generalizable this mechanism is to other thalamic nuclei. We have used whole cell recording to examine the electrophysiological properties of neurons of the anterodorsal thalamic nucleus, a nucleus thought not to undergo spindle oscillations. We now report that cells in this nucleus also display an ADP mediated by I(h). Surprisingly, the ADP and the underlying upregulation of I(h) persisted even after buffering intracellular calcium and blocking calcium influx. These results indicate that, in neurons of the anterodorsal thalamic nucleus, an I(h)-mediated ADP can occur through a mechanism that does not involve a rise in intracellular calcium. We next examined the possibility that this calcium-independent ADP might be modulated by serotonin. Serotonin produced a robust enhancement in the amplitude of the ADP even after strong buffering of intracellular calcium and blockade of calcium channels. These results indicate that neurons of the anterodorsal thalamic nucleus display a calcium-independent, I(h)-mediated ADP and that this ADP is a target for regulation by serotonin. These findings identify a novel mechanism by which serotonin can regulate neuronal excitability.

Dihydromaltophilin; a novel fungicidal tetramic acid containing metabolite from Streptomyces sp.

A new tetramic acid containing metabolite, A90931a has been isolated from Streptomyces sp., along with a second factor (A90931b) recently described and known as maltophilin. The structures were determined from spectroscopic data of the isolates and their acetylated products. A90931a was spectroscopically identical to the previously described antibiotic TAN-883b whose structure was not reported. A90931a and A90931b exhibit fungicidal activity against the grape pathogen Plasmopara viticola. Due to its similarity to maltophilin, A90931a has been called dihydromaltophilin.

Synthesis and hypoglycemic activity of substituted 8-(1-piperazinyl)imidazo[1,2-a]pyrazines.

A series of alkyl- and halo-substituted 8-(1-piperazinyl)imidazo[1,2-a]pyrazines were prepared using two approaches, the condensation of alpha-halocarbonyl derivatives with an aminopyrazine or the oxidation-dehydration of a [(beta-hydroxyalkyl)amino]pyrazine. These imidazo[1,2-a]pyrazines were evaluated for their binding affinity to the alpha 1, alpha 2, beta 1, and beta 2 adrenergic receptors as well as their ability to lower blood glucose in insulin resistant hyperglycemic ob/ob mice. Modifications on 8-(1-piperazinyl)imidazo[1,2-a]pyrazine (4) reduced alpha 2 binding, lowered hypoglycemic potency, and showed variations in binding to the alpha 1, beta 1, and beta 2 adrenergic receptors. In addition to 4, the 2-methyl, 3-methyl, and 5-methyl 8-(1-piperazinyl)imidazo[1,2-a]pyrazines (16k, 25m, and 16f, respectively) displayed high affinity for the alpha 2 receptor and were potent hypoglycemic agents when compared to 2-amino-7,8-dihydro-4-(1-piperazinyl)-6H-thiopyrano[3,2- d]pyrimidine (MTP-1403, 2). Receptor binding was modified by use of a 4-methylpiperazine moiety which reduced alpha 1 and beta 1 binding while retaining some hypoglycemic activity. The structure-activity relationship for heterocyclic alkyl and halo substitution on biological activity is discussed.

Effects of an alpha 2-adrenoceptor antagonist on glucose tolerance in the genetically obese mouse (C57BL/6J ob/ob).

This study examines the effects of a relatively selective alpha 2-adrenoceptor antagonist, 8-(L-piperazinyl)imado-[1,2-alpha] pyrazine (compound A), and the preferential alpha 2-agonist clonidine on blood glucose, glucose tolerance, and plasma insulin levels in the C57BL/6J ob/ob mouse and its lean littermate. While clonidine raised blood glucose levels and impaired glucose tolerance, oral administration of compound A resulted in decreased blood glucose levels, as well as improved glucose tolerance in ob/ob mice. Insulin levels in ob/ob mice treated with clonidine were significantly reduced, while compound A raised insulin levels threefold and blocked the effects of clonidine when co-administered to the same animals. Clonidine-induced hyperglycemia in lean littermates was not accompanied by a decrease in insulin levels, while a small but significant increase in insulin levels was observed by compound A administration. Glycogen synthesis in diaphragm of ob/ob mice was enhanced after oral administration of compound A and was accompanied by an increase in plasma insulin levels. Concomitant treatment with a potent somatostatin analog to inhibit insulin release blocked the effects of the alpha 2-adrenoceptor antagonist, compound A. These observations suggest that the alpha 2-antagonist studied, increased plasma insulin levels with an accompanying reduction in blood glucose and an improvement in glucose tolerance in a genetic model of insulin resistance. Differential sensitivity to alpha 2-agonist in these genetically obese mice, ob/ob, was demonstrated by decreased insulin levels due to clonidine administration.

Effects on the development of offspring of female mice exposed to platinum sulfate or sodium hexachloroplatinate during pregnancy or lactation.

On d 7 or 12 of gestation or on d 2 postpartum, Swiss ICR dams were administered either (1) a single intragastric dose of Pt(SO4) at the LD1 level or dilute H2SO4 at an equivalent volume, pH, and sulfate content, or (2) a single subcutaneous dose of Na2PtCl6 or phosphate-buffered saline at an equivalent volume and pH. To differentiate prenatal from postnatal effects of the compounds on the offspring, a full cross-fostering design was employed. Rate of growth (as a function of weight gain) and gross activity of the neonates were assessed on d 8 or 13 postpartum. On d 60-65 postpartum, open-field behavior (ambulations and rearings), rotarod performance, and passive avoidance learning of the adult offspring were investigated. Exposure to Pt(SO4)2 resulted in reduced offspring weight from d 8 to 45 postpartum, whereas the major effect of Na2PtCl6 was a reduction in activity level of the offspring of mothers exposed on d 12 of gestation.