Oligonucleotide-directed peptide synthesis in a ribosome- and ribozyme-free system

Peptide bond formation by the ribosome requires 23S rRNA and its interaction with the 3′-CCA end of tRNA. To investigate the possible evolutionary development of the peptidyl transfer reaction, we tried to obtain peptide bond formation without the ribosome or rRNA simply by using a piece of tRNA—an aminoacyl-minihelix—mixed with sequence-specific oligonucleotides that contained puromycin. Peptide bond formation was detected by gel electrophoresis, TLC analysis, and mass spectrometry. Peptide synthesis depended on sequence complementarity between the 3′-CCA sequence of the minihelix and the puromycin-bearing oligonucleotide. However, proximity of the reacting species was not by itself sufficient for peptide bond formation. In addition, imidazole as a catalyst was required. Its role may be similar to the recently proposed mechanism, wherein A2451 of 23S rRNA works as a general base. Thus, peptide bond formation can be achieved with a simple, minimized system that captures the essence of an interaction seen in the ribosome.

Domains of a Transit Sequence Required for in Vivo Import in Arabidopsis Chloroplasts1

Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence.

Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli.

Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid delta pH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.

Neurocytopathic effects of beta-amyloid-stimulated monocytes: a potential mechanism for central nervous system damage in Alzheimer disease.

Growing evidence indicates that cells of the mononuclear phagocyte lineage, which includes peripheral blood monocytes (PBM) and tissue macrophages, participate in a variety of neurodestructive events and may play a pivotal role in neurodegenerative conditions such as Alzheimer disease. The present study sought to determine whether exposure of PBM to beta-amyloid peptide (A beta), the major protein of the amyloid fibrils that accumulate in the brain in Alzheimer disease, could induce cytopathic activity in these cells upon their subsequent incubation with neural tissue. PBM were incubated with A beta for 3 days, centrifuged and washed to remove traces of cell-free A beta, and then applied to organotypic cultures of rat brain for varying periods of time. By using a cell-viability assay to quantitate neurocytopathic effect, an increase in the ratio of dead to live cells was detected in cultures containing A beta-stimulated PBM versus control PBM (stimulated with either bovine serum albumin or reverse A beta peptide) as early as 3 days after coculture. The ratio of dead to live cells increased further by 10 days of coculture. By 30 days of coculture, the dead to live cell ratio remained elevated, and the intensity of neurocytopathic effect was such that large areas of brain mass dissociated from the cultures. These results indicate that stimulation of PBM with A beta significantly heightens their neurocytopathic activity and highlight the possibility that inflammatory reactions in the brain play a role in the neurodegeneration that accompanies Alzheimer disease.

Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1.

The 4.6-kb region 5'-upstream from the gene encoding a cobalt-containing and amide-induced high molecular mass-nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was found to be required for the expression of the H-NHase gene with a host-vector system in a Rhodococcus strain. Sequence analysis has revealed that there are at least five open reading frames (H-ORF1 approximately 5) in addition to H-NHase alpha- and beta-subunit genes. Deletion of H-ORF1 and H-ORF2 resulted in decrease of NHase activity, suggesting a positive regulatory role of both ORFs in the expression of the H-NHase gene. H-ORF1 showed significant similarity to a regulatory protein, AmiC, which is involved in regulation of amidase expression by binding an inducer amide in Pseudomonas aeruginosa. H-ORF4, which has been found to be uninvolved in regulation of H-NHase expression by enzyme assay for its deletion transformant and Northern blot analysis for R. rhodochrous J1, showed high similarity to transposases from insertion sequences of several bacteria. Determination of H-NHase activity and H-NHase mRNA levels in R. rhodochrous J1 has indicated that the expression of the H-NHase gene is regulated by an amide at the transcriptional level. These findings suggest the participation of H-ORF4 (IS1164) in the organization of the H-NHase gene cluster and the involvement of H-ORF1 in unusual induction mechanism, in which H-NHase is formed by amides (the products in the NHase reaction), but not by nitriles (the substrates).

Parkinson's disease and gastrointestinal dysfunctions: morphological, neurochemical and functional modifications of the enteric nervous system associated with central dopaminergic impairment

Parkinson’s disease (PD) is frequently associated with gastrointestinal (GI) symptoms, mostly represented by abdominal distension, constipation and defecatory dysfunctions. Despite GI dysfunctions have a major impact on the clinical picture of PD, there is currently a lack of information on the neurochemical, pathological and functional correlates of GI dysmotility associated with PD. Moreover, there is a need of effective and safe pharmacological therapies for managing GI disturbances in PD patients. The present research project has been undertaken to investigate the relationships between PD and related GI dysfunctions by means of investigations in an animal model of PD induced by intranigral injection of 6-hydroxydopamine (6-OHDA). The use of the 6-OHDA experimental model of PD in the present program has allowed to pursue the following goals: 1) to examine the impact of central dopaminergic denervation on colonic excitatory cholinergic and tachykininergic neuromotility by means of molecular, histomorphologic and functional approaches; 2) to elucidate the role of gut inflammation in the onset and progression of colonic dysmotility associated with PD, characterizing the degree of inflammation and oxidative damage in colonic tissues, as well as identifying the immune cells involved in the production of pro-inflammatory cytokines in the gut; 3) to evaluate the impact of chronic treatment with L-DOPA plus benserazide on colonic neuromuscular activity both in control and PD animals. The results suggest that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory cholinergic neurotransmission and an enhanced tachykininergic control, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate. These motor alterations might result from the occurrence of a condition of gut inflammation associated with central intranigral denervation. The treatment with L-DOPA/BE following central dopaminergic neurodegeneration can restore colonic motility, likely through a normalization of the cholinergic enteric neurotransmission, and it can also improve the colonic inflammation associated with central dopaminergic denervation.

Steric and mass-induced sea level variations in the Mediterranean Sea revisited

The total sea level variation (SLV) is the combination of steric and mass␣induced SLV, whose exact shares are key to understanding the oceanic response to climate system changes. Total SLV can be observed by radar altimetry satellites such as TOPEX/POSEIDON and Jason 1/2. The steric SLV can be computed through temperature and salinity profiles from in situ measurements or from ocean general circulation models (OGCM), which can assimilate the said observations. The mass-induced SLV can be estimated from its time-variable gravity (TVG) signals. We revisit this problem in the Mediterranean Sea estimating the observed, steric, and mass-induced SLV, for the latter we analyze the latest TVG data set from the GRACE (Gravity Recovery and Climate Experiment) satellite mission launched in 2002, which is 3.5 times longer than in previous studies, with the application of a two-stage anisotropic filter to reduce the noise in high-degree and -order spherical harmonic coefficients. We confirm that the intra-annual total SLV are only produced by water mass changes, a fact explained in the literature as a result of the wind field around the Gibraltar Strait. The steric SLV estimated from the residual of “altimetry minus GRACE” agrees in phase with that estimated from OGCMs and in situ measurements, although showing a higher amplitude. The net water fluxes through both the straits of Gibraltar and Sicily have also been estimated accordingly.

The masses of the neutron and donor star in the high-mass X-ray binary IGR J18027-2016

Aims. We report near-infrared observations of the supergiant donor to the eclipsing high mass X-ray binary pulsar IGR J18027-2016. We aim to determine its spectral type and measure its radial velocity curve and hence determine the stellar masses of the components. Methods. ESO/VLT observations of the donor utilising the NIR spectrograph ISAAC were obtained in the H and K bands. The multi-epoch H band spectra were cross-correlated with RV templates in order to determine a radial solution for the system. Results. The spectral type of the donor was confirmed as B0-1 I. The radial velocity curve constructed has a semi-amplitude of 23.8 ± 3.1 km s-1. Combined with other measured system parameters, a dynamically determined neutron star mass of 1.4  ±  0.2–1.6  ±  0.3 M⊙ is found. The mass range of the B0-B1 I donor was 18.6  ±  0.8–21.8  ±  2.4 M⊙. These lower and upper limits were obtained under the assumption that the system is viewed edge-on (i = 90° with β = 0.89) for the lower limit and the donor fills its Roche lobe (β = 1 with i = 73.1°) for the upper limit respectively.

The evolution and masses of the neutron star and donor star in the high mass X-ray binary OAO 1657−415

We report near-infrared radial velocity (RV) measurements of the recently identified donor star in the high mass X-ray binary (HMXB) system OAO 1657−415 obtained in the H band using ISAAC on the Very Large Telescope. Cross-correlation methods were employed to construct a RV curve with a semi-amplitude of 22.1 ± 3.5 km s−1. Combined with other measured parameters of this system it provides a dynamically determined neutron star (NS) mass of 1.42 ± 0.26 M⊙ and a mass of 14.3 ± 0.8 M⊙ for the Ofpe/WN9 highly evolved donor star. OAO 1657−415 is an eclipsing HMXB pulsar with the largest eccentricity and orbital period of any within its class. Of the 10 known eclipsing X-ray binary pulsars OAO 1657−415 becomes the ninth with a dynamically determined NS mass solution and only the second in an eccentric system. Furthermore, the donor star in OAO 1657−415 is much more highly evolved than the majority of the supergiant donors in other HMXBs, joining a small but growing list of HMXBs donors with extensive hydrogen depleted atmospheres. Considering the evolutionary development of OAO 1657−415, we have estimated the binding energy of the envelope of the mass donor and find that there is insufficient energy for the removal of the donor’s envelope via spiral-in, ruling out a common envelope evolutionary scenario. With its non-zero eccentricity and relatively large orbital period the identification of a definitive evolutionary pathway for OAO 1657−415 remains problematic, we conclude by proposing two scenarios which may account for OAO 1657−415 current orbital configuration.

IRAS 18357-0604 – an analogue of the galactic yellow hypergiant IRC +10420?

Context. Yellow hypergiants represent a short-lived evolutionary episode experienced by massive stars as they transit to and from a red supergiant phase. As such, their properties provide a critical test of stellar evolutionary theory, while recent observations unexpectedly suggest that a subset may explode as Type II supernovae. Aims. The galactic yellow hypergiant IRC +10420 is a cornerstone system for understanding this phase since it is the strongest post-RSG candidate known, has demonstrated real-time evolution across the Hertzsprung-Russell diagram and been subject to extensive mass loss. In this paper we report on the discovery of a twin of IRC +10420 - IRAS 18357-0604. Methods. Optical and near-IR spectroscopy are used to investigate the physical properties of IRAS 18357-0604 and also provide an estimate of its systemic velocity, while near- to mid-IR photometry probes the nature of its circumstellar environment. Results. These observations reveal pronounced spectral similarities between IRAS 18357-0604 and IRC +10420, suggesting comparable temperatures and wind geometries. IR photometric data reveals a similarly dusty circumstellar environment, although historical mass loss appears to have been heavier in IRC +10420. The systemic velocity implies a distance compatible with the red supergiant-dominated complex at the base of the Scutum Crux arm; the resultant luminosity determination is consistent with a physical association but suggests a lower initial mass than inferred for IRC +10420 (≲20 M⊙ versus ~40 M⊙). Evolutionary predictions for the physical properties of supernova progenitors derived from ~18–20 M⊙ stars – or ~12–15 M⊙ stars that have experienced enhanced mass loss as red supergiants – compare favourably with those of IRAS 18357-0604, which in turn appears to be similar to the the progenitor of SN2011dh; it may therefore provide an important insight into the nature of the apparently H-depleted yellow hypergiant progenitors of some Type IIb SNe.

The influence of the sample introduction system on signals of different tin compounds in inductively coupled plasma-based techniques

The influence of the sample introduction system on the signals obtained with different tin compounds in inductively coupled plasma (ICP) based techniques, i.e., ICP atomic emission spectrometry (ICP–AES) and ICP mass spectrometry (ICP–MS) has been studied. Signals for test solutions prepared from four different tin compounds (i.e., tin tetrachloride, monobutyltin, dibutyltin and di-tert-butyltin) in different solvents (methanol 0.8% (w/w), i-propanol 0.8% (w/w) and various acid matrices) have been measured by ICP–AES and ICP–MS. The results demonstrate a noticeable influence of the volatility of the tin compounds on their signals measured with both techniques. Thus, in agreement with the compound volatility, the highest signals are obtained for tin tetrachloride followed by di-tert-butyltin/monobutyltin and dibutyltin. The sample introduction system exerts an important effect on the amount of solution loading the plasma and, hence, on the relative signals afforded by the tin compounds in ICP–based techniques. Thus, when working with a pneumatic concentric nebulizer, the use of spray chambers affording high solvent transport efficiency to the plasma (such as cyclonic and single pass) or high spray chamber temperatures is recommended to minimize the influence of the tin chemical compound. Nevertheless, even when using the conventional pneumatic nebulizer coupled to the best spray chamber design (i.e., a single pass spray chamber), signals obtained for di-tert-butyltin/monobutyltin and dibutyltin are still around 10% and 30% lower than the corresponding signal for tin tetrachloride, respectively. When operating with a pneumatic microconcentric nebulizer coupled to a 50 °C-thermostated cinnabar spray chamber, all studied organotin compounds provided similar emission signals although about 60% lower than those obtained for tin tetrachloride. The use of an ultrasonic nebulizer coupled to a desolvation device provides the largest differences in the emission signals, among all tested systems.

GU Monocerotis: A high-mass eclipsing overcontact binary in the young open cluster Dolidze 25

Context. The eclipsing binary GU Mon is located in the star-forming cluster Dolidze 25, which has the lowest metallicity measured in a Milky Way young cluster. Aims. GU Mon has been identified as a short-period eclipsing binary with two early B-type components. We set out to derive its orbital and stellar parameters. Methods. We present a comprehensive analysis, including B and V light curves and 11 high-resolution spectra, to verify the orbital period and determine parameters. We used the stellar atmosphere code FASTWIND to obtain stellar parameters and create templates for cross-correlation. We obtained a model to fit the light and radial-velocity curves using the Wilson-Devinney code iteratively and simultaneously. Results. The two components of GU Mon are identical stars of spectral type B1 V with the same mass and temperature. The light curves are typical of an EW-type binary. The spectroscopic and photometric analyses agree on a period of 0.896640 ± 0.000007 d. We determine a mass of 9.0 ± 0.6 M⊙ for each component and for temperatures of 28 000 ± 2000 K. Both values are consistent with the spectral type. The two stars are overfilling their respective Roche lobes, sharing a common envelope and, therefore the orbit is synchronised and circularised. Conclusions. The GU Mon system has a fill-out factor above 0.8, containing two dwarf B-type stars on the main sequence. The two stars are in a very advanced stage of interaction, with their extreme physical similarity likely due to the common envelope. The expected evolution of such a system very probably leads to a merger while still on the main sequence.

A system of ethicks: Of morall phylosophy in generall & in speciall

This hardcover volume contains manuscript copies of Charles Morton's "A System of Ethicks," "Pneumatics. Or a treatise of the Rev'd Mr. Charles Morton about ye Nature of Spirit," "Appendix of the Souls of Brutes," "Some Theological Questions Answd," and a one-page list "Texts of Scripture to prove if ye head of Christ &c." copied by Harvard student Ebenezer Williams in February 1707/8.

Vitamin D in patients with atherosclerosis : a link between atherosclerosis and bone mass ?

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Characterization of Endoproteolytic Processing of Dynorphins by Proprotein Convertases using Mouse Spinal Cord S9 Fractions and Mass Spectrometry

Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous Big Dynorphin (BDyn) and Dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild type (WT), PC1-/+ and PC2-/+ animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments Dynorphin 1-19, Dynorphin 1-13, Dynorphin 1-11 and Dynorphin 1-7 and C-terminal processing of Dyn A generates Dynorphin 1-13, Dynorphin 1-11 and Dynorphin 1-7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even partial inhibition of PC1 or PC2 may impair the endogenous opioid system.