Messenger Species Research Articles

Surface Action Spectroscopy: A Review and a Perspective on a New Technique to Study Vibrations at Surfaces.

A new vibrational spectroscopy method aimed at the investigation of solid surfaces in ultrahigh vacuum, called "Surface Action Spectroscopy (SAS)", is described and the first results are reviewed. This technique is based on ideas and experiments performed in the gas phase. A surface is exposed to a messenger species at low temperature. This messenger species is desorbed via absorption of tunable infrared light from a free-electron laser and the desorption rate of the messenger species is recorded via mass spectrometry. It is shown that the technique is extremely surface sensitive and we discuss the basic mechanisms of the technique. We show a feasibility study on a V2 O3 (0001) surface, where we know the surface structure. We then proceed to the example of iron oxide films to study the surface structure in parallel with calculations of the surface phonons, which allow us to confirm the surface structure of Fe3 O4 (111) to be Fetet terminated. It also provides evidence for the so-called biphase structure. To conclude, we discuss possibilities to apply the technique to interesting questions in model and real catalysis, since the technique may provide interesting information independent of long-range order of the sample.

CBASS Immunity Uses CARF-Related Effectors to Sense 3′–5′- and 2′–5′-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection

cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.

Minimal Yukawa deflection of AMSB from the Kahler potential

We propose a minimal Yukawa deflection scenario of AMSB from the Kahler potential through the Higgs-messenger mixing. Salient features of this scenario are discussed and realistic MSSM spectrum can be obtained. Such a scenario, which are very predictive, can solve the tachyonic slepton problem with less messenger species. Numerical results indicate that the LOSPs predicted by this scenario can not be good DM candidates. So it is desirable to extend this scenario with a Peccei-Quinn sector to solve the strong CP problem and at the same time provide new DM candidates. We propose a way to obtain a light axino mass in SUSY KSVZ axion model with Yukawa deflected anomaly mediation SUSY breaking mechanism. The axino can possibly be the LSP and act as a good DM candidate.

Radiative natural SUSY spectrum from deflected AMSB scenario with messenger-matter interactions

A radiative natural SUSY spectrum are proposed in the deflected anomaly mediation scenario with general messenger-matter interactions. Due to the contributions from the new interactions, positive slepton masses as well as a large |A_t| term can naturally be obtained with either sign of deflection parameter and few messenger species (thus avoid the possible Landau pole problem). In this scenario, in contrast to the ordinary (radiative) natural SUSY scenario with under-abundance of dark matter (DM), the DM can be the mixed bino-higgsino and have the right relic density. The 125 GeV Higgs mass can also be easily obtained in our scenario. The majority of low EW fine tuning points can be covered by the XENON-1T direct detection experiments.

750 GeV diphoton resonance, 125 GeV Higgs and muon g− 2 anomaly in deflected anomaly mediation SUSY breaking scenarios

We propose to interpret the 750 GeV diphoton excess in deflected anomaly mediation supersymmetry breaking scenarios, which can naturally predict couplings between a singlet field and vector-like messengers. The CP-even scalar component (S) of the singlet field can serve as the 750 GeV resonance. The messenger scale, which is of order the gravitino scale, can be as light as F_\phi \sim {\cal O}(10) TeV when the messenger species N_F and the deflection parameter d are moderately large. Such messengers can induce the large loop decay process S \to \gamma\gamma. Our results show that such a scenario can successfully accommodate the 125 GeV Higgs boson, the 750 GeV diphoton excess and the muon g-2 without conflicting with the LHC constraints. We also comment on the possible explanations in the gauge mediation supersymmetry breaking scenario.

Deflected anomaly mediated SUSY breaking scenario with general messenger–matter interactions

We propose to introduce general messenger–matter interactions in the deflected anomaly mediated SUSY breaking scenario. The most general form for the resulting soft parameters is derived. New interference terms between the GMSB type and AMSB type contributions are the unique feature of this scenario. Messenger–matter interactions involving sleptons can be used to solve the tachyonic slepton problem and naturally lead to positive slepton masses regardless of the sign of deflection parameter. Besides, due to the new contributions, large |At| that will not trigger color-breaking stop VEV are also possible in this scenario, thus can easily give the 125 GeV higgs which was discovered by LHC. This type of deflected AMSB scenario need very few messenger species, thus can avoid possible non-perturbative gauge couplings below the GUT scale (or Landau pole below the Planck scale).

Electrochemical Measurement of Endogenous Serotonin Release from Human Blood Platelets

Platelet aggregation in the bloodstream is tightly associated with the secretory function of platelets based on several types of cytoplasmic secretory granules, each sequestering distinct chemical messenger species. Dense-body granules are one prominent type of secretory granule responsible for storing small molecule chemical messengers. Upon platelet activation, the timely and rapid release of these small molecules is critical in facilitating platelet aggregation. Therefore, techniques capable of measuring real-time granule content release are needed to understand the fundamental properties of platelet secretion and aggregation. Existing techniques lack adequate time resolution or require potentially toxic exogenous reagents for real-time measurement of granule content release. Herein, we demonstrate a label-free electrochemical method based on the endogenous electroactive chemical messenger serotonin (5-hydroxytryptamine or 5-HT) for the real-time measurement of dense-body granule secretion from platelet suspensions; fast-scan cyclic voltammetry (FSCV) using carbon-fiber microelectrodes was chosen on the basis of its excellent temporal resolution, high sensitivity, and the ability to provide the electrochemical signature cyclic voltammograms for molecular identification. Real-time serotonin release from thrombin-stimulated human platelet suspensions was successfully measured, and the amount and time course of the bulk serotonin release were found to agree well with data obtained from single platelet measurements, thus confirming accurate characterization of granular secretion. Furthermore, this electrochemical method was applied to study the stimulation-secretion coupling in platelets, serotonin storage and release dynamics with applied pharmacological agents, and chemical messenger storage deficiency in Hermansky-Pudlak Syndrome (HPS) platelets, and the potential of this method to reveal secretion behavior in both normal and diseased platelets has clearly been demonstrated.

Synchronization of Chemical Micro-oscillators

Many phenomena of biological, physical, and chemical importance involve synchronization of oscillatory elements. We explore here, in several geometries, the behavior of diffusively coupled, nanoliter volume, aqueous drops separated by oil gaps and containing the reactants of the oscillatory Belousov−Zhabotinsky (BZ) reaction. A variety of synchronous regimes are found, including in- and antiphase oscillations, stationary Turing patterns, and more complex combinations of stationary and oscillatory BZ drops, including three-phase patterns. A model consisting of ordinary differential equations based on a simplified description of the BZ chemistry and diffusion of messenger (primarily inhibitory) species qualitatively reproduces most of the experimental results.

Primary afferent second messenger cascades interact with specific integrin subunits in producing inflammatory hyperalgesia

We recently reported that hyperalgesia induced by the inflammatory mediator prostaglandin E 2 (PGE 2) requires intact α1, α3 and β1 integrin subunit function, whereas epinephrine-induced hyperalgesia depends on α5 and β1. PGE 2-induced hyperalgesia is mediated by protein kinase A (PKA), while epinephrine-induced hyperalgesia is mediated by a combination of PKA, protein kinase Cε (PKCε) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). We hypothesized that inflammatory mediator-induced hyperalgesia involves specific interactions between different subsets of integrin subunits and particular second messenger species. In the present study, function-blocking anti-integrin antibodies and antisense oligodeoxynucleotides were used to elucidate these interactions in rat. Hyperalgesia produced by an activator of adenylate cyclase (forskolin) depended on α1, α3 and β1 integrins. However, hyperalgesia induced by activation of the cascade at a point farther downstream (by cAMP analog or PKA catalytic subunit) was independent of any integrins tested. In contrast, hyperalgesia induced by a specific PKCε agonist depended only on α5 and β1 integrins. Hyperalgesia induced by agonism of MAPK/ERK depended on all four integrin subunits tested (α1, α3, α5 and β1). Finally, disruption of lipid rafts antagonized hyperalgesia induced by PGE 2 and by forskolin, but not that induced by epinephrine. Furthermore, α1 integrin, but not α5, was present in detergent-resistant membrane fractions (which retain lipid raft components). These observations suggest that integrins play a critical role in inflammatory pain by interacting with components of second messenger cascades that mediate inflammatory hyperalgesia, and that such interaction with the PGE 2-activated pathway may be organized by lipid rafts.

Tissue-specific expression of two structurally different estrogen receptor alpha isoforms along the female reproductive axis of an oviparous species, the rainbow trout.

In oviparous species, in addition to a full-length estrogen receptor alpha (ER alpha), another ER alpha isoform lacking the A domain and exhibiting a ligand-independent transactivation function has been consistently reported. Although both isoforms are expressed in the liver, their respective sites of expression in other potential target tissues are unknown. In contrast to the situation in Xenopus and chicken, the two isoforms of rainbow trout (Oncorhynchus mykiss) are generated from two classes of transcripts with different 5' untranslated sequences issued from the same gene by alternative splicing and promoter usage. The aim of this study was to take advantage of the unique organization of the rainbow trout ER alpha gene to investigate the tissue distribution of these two messenger species along the reproductive axis of female trout. The S1 nuclease assay and in situ hybridization were used, with probes specific for each of the transcripts. Reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the isoforms also was performed. The data indicated that the full-length ER alpha is expressed in liver, brain, pituitary, and ovary, whereas expression of the isoform with the truncated A domain is restricted to the liver, demonstrating a tissue-specific expression of these two ER alpha isoforms. The presence of a short liver-specific isoform in oviparous species suggests its role in the development and/or maintenance of the unique function of the liver in the vitellogenesis process.

A study of selected Plasmodium yoelii messenger RNAs during hepatocyte infection

We investigated the expression of several mRNAs in exoerythrocytic and erythrocytic stages of Plasmodium yoelii in infected mice, focusing our attention on genes thought to be involved in signal transduction (like pypka and pymap- 1, encoding homologues of cAMP-dependent and mitogen-activated protein kinases, respectively) and cell cycle progression (those encoding the cdc2-related kinases Pycrk-1, Pycrk-3 and Pymrk). Messengers coding for enzymes involved in general processes such as DNA replication and RNA transcription (both subunits of the ribonucleotide reductase (Pyrnr1, Pyrnr2) and RNA polymerase II) as well as a messenger coding for Pys21, a sexual stage-specific protein, were also investigated. Total RNA was prepared from livers of infected mice at different times post sporozoite inoculation. In contrast to the pys21 transcript, which was observed only in infected erythrocytes, all messenger species could be detected in the liver by RT-PCR, peaking at 43 h post infection, a time when parasite burden was maximum, and decreasing markedly thereafter to become hardly visible at 168 h. Some transcripts ( pypka, pymap-1, pyrnr1 and pyrnr2) could be detected 12 h after infection, while others ( pymrk and pyrnapolII) did not become detectable until 24 h. In addition, we characterised all these messengers by Northern blot of total RNAs extracted from infected erythrocytes. Taken together, these data suggest that a similar set of regulatory genes is expressed during both exoerythrocytic and erythrocytic schizogony.

Cloning of a rat brain succinic semialdehyde reductase involved in the synthesis of the neuromodulator gamma-hydroxybutyrate.

The gamma-hydroxybutyrate biosynthetic enzyme succinic semialdehyde reductase (SSR) was purified to homogeneity from rat brain. Peptides were generated by tryptic cleavage and sequenced. PCR primers were designed from the amino acid sequences of two of the peptides showing a similarity (75-85%) to a mitochondrial aldehyde dehydrogenase. A PCR-amplified DNA fragment was generated from recombinant plasmids prepared by a mass excision procedure from a rat hippocampal cDNA library and used as a probe to screen this cDNA library. One cDNA of 1341 bp had an open reading frame encoding a protein of 447 residues with a deduced molecular mass of 47967 Da. The enzyme was expressed in Escherichia coli. Immunoblotting analysis revealed the existence of a protein with the same electrophoretic mobility as the SSR purified from rat brain and with an estimated molecular mass of 45 kDa. Northern blot experiments showed that this enzyme was not expressed in the kidney or in the liver. In the brain tissue, a single but rather broad band was labelled under high stringency conditions, suggesting the presence of more than one messenger species coding for SSR. Hybridization in situ performed on brain tissue slices showed specific labelling of the hippocampus, the upper cortex layer, the thalamus, the substantia nigra, the cerebellum, the pons medulla and the olfactory tract. The recombinant enzyme showed catalytic properties similar to those of the SSR purified from rat brain, particularly in regard to its substrate affinities and Ki for inhibition by phthalaldehydic acid. Valproic acid did not inhibit the cloned SSR. This enzyme had 20-35% identity in highly conserved regions involved in NADPH binding with four other proteins belonging to the aldo-oxo reductase family.

Localization and sequence analysis of poly(A) sites generating multiple dihydrofolate reductase mRNAs.

The murine dihydrofolate reductase (DHFR) gene gives rise to multiple polyadenylated mRNAs displaying heterogeneity in the length of the 3' untranslated region. These species are present in the cytoplasm at levels that vary over 2 orders of magnitude, suggesting that certain poly(A) sites are preferred over others. Previous observations have shown that three out of the four major sites of polyadenylation do not display consensus hexanucleotide (AATAAA, ATTAAA) signals. We have further analyzed the sequences involved in directing multiple polyadenylation events on the DHFR gene by focusing our attention on the 4.1- and 5.6-kilobase mRNAs, the lowest abundance DHFR species observed on RNA blot analysis. Identification and sequence analysis of the poly(A) addition sites corresponding to these species revealed appropriately positioned consensus hexanucleotide signals; additional nearby poly(A) sites were also detected which apparently do not use consensus hexanucleotides to direct poly(A) addition to DHFR mRNAs of relatively lower abundance. We have also identified polyadenylation sites downstream of the 4.1- and 5.6-kilobase sites which display consensus hexanucleotide signals and correspond to messenger species too rare for detection by routine RNA blot analysis. Our data bring to 11 the number of known functional poly(A) addition sites associated with the DHFR gene.

Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen.

Three cDNA clones, corresponding to two non-overlapping regions of the mRNA coding for the mouse p53 cellular tumor antigen, were isolated and characterized. In hybridization-selection assays, these clones were capable of selectively binding p53 mRNA, as demonstrated by in vitro translation and immunoprecipitation with anti-p53 monoclonal antibodies. The p53 mRNA appeared to be the only messenger species specifically selected by these clones. The size of the p53 mRNA was found to be approximately 2 kb, and its levels to vary substantially among different types of transformed cells. Evidence was found for the existence of two distinct p53-specific genes in mouse genomic DNA. Two partially overlapping recombinant phage clones were obtained, both derived from the same p53-specific genomic DNA region. The orientation of the various cDNA clones relative to that of the p53 mRNA was established by S1 analysis and the relationship between the cDNA clones and the genomic ones was determined by comparative restriction enzyme mapping and nucleic acid hybridization.

Molecular cloning of the cDNA coding for rat ornithine transcarbamoylase.

Ornithine transcarbamoylase is a mitochondrial matrix enzyme composed of three identical subunits encoded on the X chromosome. The subunit is synthesized on cytoplasmic polysomes as a precursor that is cleaved during transport into mitochondria. We report here the isolation and characterization of cDNA clones containing sequences corresponding to the mRNA encoding the ornithine transcarbamoylase subunit. cDNA was synthesized using rat liver mRNA enriched by polysome immunoadsorption for the low-abundance messenger species encoding the enzyme subunit. After insertion of cDNA into plasmid pBR322 and cloning in Escherichia coli, identification of the desired plasmids was accomplished by (i) differential colony hybridization using cDNA probes synthesized from mRNA of various tissues; (ii) differential blot hybridization using cDNA probes synthesized from mRNA enriched for or depleted of the ornithine transcarbamoylase message; (iii) hybrid-selected translation assays; and (iv) most definitively, structural analysis, which matched 25 consecutive amino acid residues determined by sequential Edman analysis of the carboxyl-terminal portion of the purified enzyme subunit with coding sequence present in the insert of one of the plasmids.

Epidermal keratin messenger RNAs: A heterogeneous family

A messenger RNA fraction from guinea-pig skin sediments on sucrose gradients at approx. 19 S and codes for keratin polypeptides in a messenger-dependent reticulocyte lysate system. Partial purification of this fraction was achieved by two cycles of chromatography on oligo(dT)-cellulose, followed by two cycles of sucrose gradient centrifugation. The identities of the protein products as keratins were established by co-electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, by peptide mapping, and by co-electrophoresis on two-dimensional polyacrylamide gels. All of the epidermal keratin polypeptides which are present in vivo are synthesized in vitro under the direction of this messenger. Fractionation of the messenger indicates that each different polypeptide is the product of a single mRNA species, and that no keratin is formed by proteolytic processing of higher molecular weight species or by polymerization of smaller precursors. Post-translational changes such as phosphorylation, which are known to occur in vivo, cannot be identified in the reticulocyte lysate system. Translation of these keratin messenger species is strongly inhibited by 7-methylguanosine 5′-triphosphate, indicating that the molecules have a ‘capped’ 5′-terminus.

Identification and initial characterization of a new low-molecular-weight virus-encoded T antigen in a line of simian virus 40-transformed cells.

SV80 cells, a simian virus 40 (SV40)-transformed derivative of a strain of human fibroblasts, synthesize an 8-kilodalton anti-T reactive polypeptide in addition to large T and small t antigens. Although not observed during lytic infection carried out under a variety of conditions, an anti-T reactive molecule which comigrated with the SV80 8-kilodalton protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis was synthesized by one of five other SV40-transformed cell lines studied. The SV40 8-kilodalton protein was present in lysates of cells exposed to a brief pulse of radioactive methionine and did not accumulate during an extended chase period. This polypeptide could not by generated by mixing an unlabeled extract of SV80 cells with a labeled extract of infected monkey cells. The 8-kilodalton molecule reacts with antibody raised against homogeneous large T antigen, is present only in the cytoplasm, is not complexed with T, lacks DNA-binding properties, and is not phosphorylated. This protein could be translated in a cell-free system programmed by SV40-specific mRNA. At least two messenger species (approximately 19S and approximately 22S) directed its synthesis. Tryptic peptide analysis of [35S]methionine-labeled proteins demonstrated that the 8-kilodalton protein contains all eight of the common T/t peptides and one additional peptide not present in the maps of t or T. It lacks both of the t-unique peptides. The organization of the integrated viral sequences which encode this molecule was determined by restriction endonuclease analysis. In particular, SV80 cells contain at least two integrated SV40 genomes which are oriented in tandem, with an intervening cellular sequence..

The interaction of human haemoglobin with allosteric effectors as a model for drug-receptor interactions.

1 The release of bound oxygen from oxyhaemoglobin by allosteric effectors is considered as a model for those drug-receptor interactions where the primary response to agonist binding is the release of a second messenger species. 2 A theory of haemoglobin oxygenation, based on the two-state model of Monod, Wyman & Changeux (1965) is used to predict the relationship between 'pharmacological' response and dose of agonist. This relationship is the same as that derived from classical pharmacological occupancy theory. 3 The potency of an agonist is a weighted average of its affinities for the two conformational states of the receptor. 4 The efficacy of an agonist depends not only upon its binding to one of the two conformational states, but also on its ability to alter the functional properties of that state by lowering the affinity of the state for the second messenger. 5 2,3-Diphosphoglycerate and adenosine triphosphate are approximately equipotent and of similar efficacy, but inositol hexaphosphate is about 500 times more potent and has a higher efficacy.

Sequences of two 5′-terminal ribosome-protected fragments from reovirus messenger RNAs

Wheat germ ribosomes can bind to reovirus messenger RNAs, protecting a limited region of each message against digestion by T 1 RNAase. From the medium size class of reovirus mRNA, which includes three messenger species, three protected fragments were recovered from 40 S initiation complexes; and each of the fragments included the 5′-terminal methylated “cap”, m 7 G(5′)-ppp(5′)G m -C. Two of the 40 S ribosome-protected fragments, consisting of 52 and 44 nucleotides, were sequenced. Each of these fragments contained an AUG codon and was capable of re-binding to ribosomes. The two ribosome-protected fragments showed limited sequence homology. Two of the fragments recovered from 80 S initiation complexes with the medium size class of reovirus RNA were also sequenced. The sequences of each message protected by 80 S ribosomes were a subset of those protected by 40 S ribosomes. With the two messages studied in greatest detail, 80 S ribosomes failed to protect the 5′-terminal region of either message, and in one case, the 3′-terminal portion of the fragment protected by 40 S ribosomes also was not protected within 80 S initiation complexes.

Control of protein synthesis in human fibroblasts by intracellular potassium.

Abstract Intracellular potassium ion (K+) in cultured human fibroblasts (HF cells) was maintained at reduced steady-state levels by incubating cells in various ouabain concentrations. Small decreases in cell K+ had no effect on protein synthesis and cell growth, but when cell K+ fell below 60–80% of control levels, the rate of protein synthesis decreased in proportion to further reductions in K+. DNA synthesis was also inhibited, presumably because of its dependence on protein synthesis. On the other hand, RNA synthesis remained uninhibited over a wide range of K+ concentrations, an effect characteristic of many specific inhibitors of protein synthesis. In ouabain-treated cells neither levels of ATP nor transport of amino acids was limiting for protein synthesis. Loss of activity of messenger or other species of RNA was not responsible for inhibition of protein synthesis, since in the presence of actinomycin D, the rate of protein synthesis could be decreased or increased solely by adjusting cell K+. Release from ouabain inhibition restored K+ levels, macromolecular synthesis, and cell growth, but there was no resulting synchrony of cell division. In cell populations partially synchronized by serum starvation and refeeding protein synthesis was sensitive to reduction in K+ levels throughout the cell cycle. Our quantitative results show that cell K+ levels, when sufficiently reduced, can determine the rate of protein synthesis and hence the rate of cell growth.