In Vivo Activation of Azipropofol Prolongs Anesthesia and Reveals Synaptic Targets

General anesthetic photolabels have been instrumental in discovering and confirming protein binding partners and binding sites of these promiscuous ligands. We report the in vivo photoactivation of meta-azipropofol, a potent analog of propofol, in Xenopus laevis tadpoles. Covalent adduction of meta-azipropofol in vivo prolongs the primary pharmacologic effect of general anesthetics in a behavioral phenotype we termed “optoanesthesia.” Coupling this behavior with a tritiated probe, we performed unbiased, time-resolved gel proteomics to identify neuronal targets of meta-azipropofol in vivo. We have identified synaptic binding partners, such as synaptosomal-associated protein 25, as well as voltage-dependent anion channels as potential facilitators of the general anesthetic state. Pairing behavioral phenotypes elicited by the activation of efficacious photolabels in vivo with time-resolved proteomics provides a novel approach to investigate molecular mechanisms of general anesthetics.     

Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239Δnef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients

The live attenuated simian immunodeficiency virus (SIV) SIVmac239Δnef is the most effective SIV/human immunodeficiency virus (HIV) vaccine in preclinical testing. An understanding of the mechanisms responsible for protection may provide important insights for the development of HIV vaccines. Leveraging the uniquely restricted genetic diversity of Mauritian cynomolgus macaques, we performed adoptive transfers between major histocompatibility complex (MHC)-matched animals to assess the role of cellular immunity in SIVmac239Δnef protection. We vaccinated and mock vaccinated donor macaques and then harvested between 1.25 × 10⁹ and 3.0 × 10⁹ mononuclear cells from multiple tissues for transfer into 12 naive recipients, followed by challenge with pathogenic SIVmac239. Fluorescently labeled donor cells were detectable for at least 7 days posttransfer and trafficked to multiple tissues, including lung, lymph nodes, and other mucosal tissues. There was no difference between recipient macaques'' peak or postpeak plasma viral loads. A very modest difference in viral loads during the chronic phase between vaccinated animal cell recipients and mock-vaccinated animal cell recipients did not reach significance (P = 0.12). Interestingly, the SIVmac239 challenge virus accumulated escape mutations more rapidly in animals that received cells from vaccinated donors. These results may suggest that adoptive transfers influenced the course of infection despite the lack of significant differences in the viral loads among animals that received cells from vaccinated and mock-vaccinated donor animals.     

Ocean energy and the law of the sea: The need for a protocol

Although fossil fuels are the overwhelming source of energy for the world, and will continue to be so for the foreseeable future, demographic, environmental, political, and economic factors indicate that interest in alternative, renewable sources of energy will grow. There is a need for both global and national policies on ocean energy management. In particular, coastal states and the energy industry would benefit from guidelines that helped to create a predictable, stable environment in which long‐term, high‐cost research, development, and investment decisions could be made with confidence. Coastal states have jurisdiction over the maritime zones most relevant to energy production, but many lack the expertise and funds to develop this potential source. Industry must operate within the control of coastal states and will not be served well by a plethora of differing legal interpretations and unilaterally imposed restrictions and obligations from state to state. An Ocean Energy Protocol to the 1982 UN Convention on the Law of the Sea would afford governments and industry the opportunity to clarify their respective obligations and address particular interests for mutual benefit.     

Beguiling and risky: ‘environmental works and measures’ for wetland conservation under a changing climate

In Australia’s Murray–Darling Basin, small-scale engineering works called ‘environmental works and measures’ have been implemented as a basis for river and other wetland conservation. While implementing these, governments seem to have embraced the beguiling notion that scarce water supplies can be divided further, while conserving the environment and maintaining agricultural production. The difficulties in doing this are expected to increase in the face of extreme climate variability. With this scenario as a backdrop, the $280 million (Monetary values ($) in this paper are in Australian dollars (AUD). At the time of writing AUD $1.00 = ~USD $1.02.) Living Murray and related programmes are assessed to see whether microengineering works to manage the hydrology of wetlands make for effective adaptation to water scarcity and climate change or whether it amounts to an overly narrow adaptation or maladaptation. Some measures were found to be substantially beneficial, such as the construction of fishways. However, under these programmes, only 0.6% of the Basin’s wetlands would be inundated and there are significant risks including desiccation of non-target wetlands and further reductions in water allocations for the environment. It is recommended that trade-offs between alternative strategies are assessed as the basis for minimising perverse impacts under changing climatic and hydrological conditions.     

Dimeric argininamide-type neuropeptide Y receptor antagonists: Chiral discrimination between Y1 and Y4 receptors

The structurally related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are endogenous agonists of the NPY receptor (YR) family, which in humans comprises four functionally expressed subtypes, designated Y₁R, Y₂R, Y₄R and Y₅R. Nonpeptide antagonists with high affinity and selectivity have been described for the Y₁R, Y₂R and Y₅R, but such compounds are still lacking for the Y₄R. In this work, the structures of the high affinity selective (R)-argininamide-type Y₁R antagonists BIBP3226 and BIBO3304 were linked via the guanidine or urea moieties to give homo-dimeric argininamides with linker lengths ranging from 31 to 41 atoms. Interestingly, the twin compounds proved to be by far less selective for the Y₁R than the R-configured monovalent parent compounds. The decrease in selectivity ratio was most pronounced for Y₁R versus Y₄R subtype, resulting in comparable affinities of bivalent ligands for Y₁R and Y₄R (e.g. UR-MK177 ((R,R)-49): K_i = 230 nM (Y₁R) and 290 nM (Y₄R)). With a K_i value of 130 nM and a K_b value of 20 nM, UR-MK188 ((R,R)-51) was superior to all Y₄R antagonists known to date. The S,S-configured optical antipodes of UR-MK177 and UR-MK188 (UR-MEK381 ((S,S)-49) and UR-MEK388 ((S,S)-51)) were synthesized to investigate the stereochemical discrimination by the different receptor subtypes. Whereas preference for R,R-configured argininamides was characteristic of the Y₁R, stereochemical discrimination by the Y₄R was not observed. This may pave the way to selective Y₄R antagonists.     

On the migration of the Whimbrel,Numenius phaeopus,in Turkey

Abstract

The Whimbrel (Numenius phaeopus) is a scarce but regular passage visitor in Turkey. Spring migration has its peak in April, autumn migration in August/September (medians are April 23th and August 29th). Detailed information on flock size, wintering, main resting sites etc. is given and compared with other Middle East countries.     

Insight into the molecular recognition of spermine by DNA quadruplexes from an NMR study of the association of spermine with the thrombin‐binding aptamer

The preferred residence sites and the conformation of DNA‐bound polyamines are central to understanding the regulatory roles of polyamines. To this end, we have used a series of selective ¹³C‐edited and selective total correlation spectroscopy‐edited one‐dimensional (1D) nuclear Overhauser effect spectroscopy NMR experiments to determine a number of intramolecular ¹H nuclear Overhauser effect (NOE) connectivities in ¹³C‐labelled spermine bound to the thrombin‐binding aptamer. The results provide evidence that the aptamer‐bound spermine adopts a conformation that optimizes electrostatic and hydrogen bond contacts with the aptamer backbone. The distance between the nitrogen atoms of the central aminobutyl is reduced by an increase in the population of gauche conformers at the C6–C7 bonds, which results in either a curved or S‐shaped spermine conformation. Molecular modelling contributes insight toward the mode of spermine binding of these spermine structures within the narrow grooves of DNA quadruplexes. In each case, the N5 ammonium group makes hydrogen bonds with two nearby phosphates across the narrow groove. Our results have implications for the understanding of chromatin structure and the rational design of quadruplex‐binding drugs. Copyright © 2013 John Wiley & Sons, Ltd.     

Barth syndrome: Cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation

Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I + III₂ + IV_n but also decreased amounts of individual complexes I and IV and supercomplexes I + III and III + IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.     

Complexation of C6-Ceramide with Cholesteryl Phosphocholine – A Potent Solvent-Free Ceramide Delivery Formulation for Cells in Culture

Ceramides are potent bioactive molecules in cells. However, they are very hydrophobic molecules, and difficult to deliver efficiently to cells. We have made fluid bilayers from a short-chain D-erythro-ceramide (C6-Cer) and cholesteryl phosphocholine (CholPC), and have used this as a formulation to deliver ceramide to cells. C6-Cer complexed with CholPC led to much larger biological effects in cultured cells (rat thyroid FRTL-5 and human HeLa cells in culture) compared to C6-Cer dissolved in dimethyl sulfoxide (DMSO). Inhibition of cell proliferation and induction of apoptosis was significantly more efficient by C6-Cer/CholPC compared to C6-Cer dissolved in DMSO. C6-Cer/CholPC also permeated cell membranes and caused mitochondrial Ca²⁺ influx more efficiently than C6-Cer in DMSO. Even though CholPC was taken up by cells to some extent (from C6-Cer/CholPC bilayers), and was partially hydrolyzed to free cholesterol (about 9%), none of the antiproliferative effects were due to CholPC or excess cholesterol. The ceramide effect was not limited to D-erythro-C6-Cer, since L-erythro-C6-Cer and D-erythro-C6-dihydroCer also inhibited cell priolifereation and affected Ca²⁺ homeostasis. We conclude that C6-Cer complexed to CholPC increased the bioavailability of the short-chain ceramide for cells, and potentiated its effects in comparison to solvent-dissolved C6-Cer. This new ceramide formulation appears to be superior to previous solvent delivery approaches, and may even be useful with longer-chain ceramides.