Microbial processes of the carbon and sulfur cycles in the Chukchi Sea

The research performed in August 2004 within the framework of the Russian-American Long-term Census of the Arctic (RUSALCA) resulted in the first data concerning the rates of the key microbial processes in the water column and bottom sediments of the Bering strait and the Chukchi Sea. The total bacterial counts in the water column varied from 30 × 10³ cells ml^?1 in the northern and eastern parts to 245 × 10³ cells ml^?1 in the southern part. The methane content in the water column of the Chukchi sea varied from 8 nmol CH₄l^?1 in the eastern part of the sea to 31 nmol CH₄l^?1 in the northern part of the Herald Canyon. Microbial activity occurred in the upper 0–3 cm of the bottom sediments; the methane formation rate varied from 0.25 to 16 nmol CH₄dm^?3 day^?1. The rates of methane oxidation varied from 1.61 to 14.7 nmol CH₄dm^?3 day^?1. The rates of sulfate reduction varied from 1.35 to 16.2 μmol SO ₄ ^2? dm^?1 day^?1. The rate of methane formation in the sediments increased with depth, while sulfate reduction rates decreased (less than 1 μmol SO ₄ ^2? dm^?3 day^?1). These high concentrations of biogenic elements and high rates of microbial processes in the upper sediment layers suggest a specific type of trophic chain in the Chukchi Sea. The approximate calculated balance of methane emission from the water column into the atmosphere is from 5.4 to 57.3 μmol CH₄m^?2 day^?1.     

Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 microM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.     

The HC fragment of tetanus toxin forms stable, concentration-dependent dimers via an intermolecular disulphide bond

Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.     

Molecular architecture and divalent cation activation of TvoK, a prokaryotic potassium channel

RCK (regulator of conductance of potassium) domains form a family of ligand-binding domains found in many prokaryotic K+ channels and transport proteins. Although many RCK domains contain an apparent nucleotide binding motif, some are known instead to bind Ca2+, which can then facilitate channel opening. Here we report on the molecular architecture and ligand activation properties of an RCK-containing potassium channel cloned from the prokaryote Thermoplasma volcanium. This channel, called TvoK, is of an apparent molecular mass and subunit composition that is consistent with the hetero-octameric configuration hypothesized for the related MthK (Methanobacterium thermoautotrophicum potassium) channel, in which four channel-tethered RCK domains coassemble with four soluble (untethered) RCK domains. The expression of soluble TvoK RCK subunits arises from an unconventional UUG start codon within the TvoK gene; silent mutagenesis of this alternative start codon abolishes expression of the soluble form of the TvoK RCK domain. Using single channel recording of purified, reconstituted TvoK, we found that the channel is activated by Ca2+ as well as Mg2+, Mn2+, and Ni2+. This non-selective divalent activation is in contrast with the activation properties of MthK, which is selectively activated by Ca2+. Transplantation of the TvoK RCK domain into MthK generates a channel that can be activated by Mg2+, illustrating that the Mg2+ binding site is likely contained within the RCK domain. We present a working hypothesis for TvoK gating in which the binding of either Ca2+ or Mg2+ can contribute approximately 5 kcal/mol toward stabilization of the open conformation of the channel.     

Fasting and glucose effects on pituitary leptin expression: is leptin a local signal for nutrient status?

Leptin, a potent anorexigenic hormone, is found in the anterior pituitary (AP). The aim of this study was to determine whether and how pituitary leptin-bearing cells are regulated by nutritional status. Male rats showed 64% reductions in pituitary leptin mRNA 24 hr after fasting, accompanied by significant (30-50%) reductions in growth hormone (GH), prolactin, and luteinizing hormone (LH), and 70-80% reductions in target cells for gonadotropin-releasing hormone or growth hormone-releasing hormone. There was a 2-fold increase in corticotropes. Subsets (22%) of pituitary cells coexpressed leptin and GH, and <5% coexpressed leptin and LH, prolactin, thyroid-stimulating hormone, or adrenocorticotropic hormone. Fasting resulted in significant (55-75%) losses in cells with leptin proteins or mRNA, and GH or LH. To determine whether restoration of serum glucose could rescue leptin, LH, and GH, additional fasted rats were given 10% glucose water for 24 hr. Restoring serum glucose in fasted rats resulted in pituitary cell populations with normal levels of leptin and GH and LH cells. Similarly, LH and GH cells were restored in vitro after populations from fasted rats were treated for as little as 1 hr in 10-100 pg/ml leptin. These correlative changes in pituitary leptin, LH, and GH, coupled with leptin's rapid restoration of GH and LH in vitro, suggest that pituitary leptin may signal nutritional changes. Collectively, the findings suggest that pituitary leptin expression could be coupled to glucose sensors like glucokinase to facilitate rapid responses by the neuroendocrine system to nutritional cues.     

Potomacanthus lobatus gen. et sp. nov., a new flower of probable Lauraceae from the Early Cretaceous (Early to Middle Albian) of eastern North America

A charcoalified fossil flower, Potomacanthus lobatus gen. et sp. nov., is described from the Early Cretaceous (Early to Middle Albian) Puddledock locality, Virginia, USA. Internal floral structure was studied using nondestructive synchrotron-radiation x-ray tomographic microscopy (SRXTM). The flower is bisexual and trimerous. The perianth consists of two whorls of tepals. The androecium has two whorls of fertile stamens. Anthers open by two distally hinged valves. The gynoecium consists of a single carpel that is plicate in the style and ascidiate in the ovary and contains a single pendant ovule. The fossil flower shares many similarities with flowers of extant Lauraceae and is unlike flowers of other families of Laurales. However, the fossil flower also differs in detail from all extant or fossil Lauraceae, particularly in configuration of the androecium. The new taxon, together with previously described but more fragmentary material from the Puddledock locality, provides the earliest fossil record of plants more closely related to Lauraceae than to any other extant family. It reveals several derived morphological characters that are potential synapomorphies among extant representatives of the family Lauraceae and contributes to the growing evidence for an early diversification of Laurales before the end of the Early Cretaceous.     

Noradrenaline modulates transmission at a central synapse by a presynaptic mechanism

The lateral division of the central amygdala (CeAL) is the target of ascending fibers from the pain-responsive and stress-responsive nuclei in the brainstem. We show that single fiber inputs from the nociceptive pontine parabrachial nucleus onto CeAL neurons form suprathreshold glutamatergic synapses with multiple release sites. Noradrenaline, acting at presynaptic alpha2 receptors, potently inhibits this synapse. This inhibition results from a decrease in the number of active release sites with no change in release probability. Introduction of a presynaptic scavenger of Gbetagamma subunits blocked the effects of noradrenaline, and botulinum toxin A reduced its effects, showing a direct action of betagamma subunits on the release machinery. These data illustrate a mechanism of presynaptic modulation where the output of a large multiple-release-site synapse is potently regulated by endogenously released noradrenaline and suggests that the CeA may be a target for the central nociceptive actions of noradrenaline.     

Ecological niche breadth and microhabitat guild structure in temperate Australian reptiles: Implications for natural resource management in endangered grassy woodland ecosystems

Ecological theory predicts that species with narrow niche requirements (habitat specialists) are more vulnerable to anthropocentric disturbances than those with broad niche requirements (habitat generalists). Hence, understanding a species ecological niche and guild membership would serve as a valuable management tool for providing a priori assessments of a species extinction risk. It also would help to forecast a species capacity to respond to land use change, as what might be expected to occur under financial incentive schemes to improve threatened ecological vegetation communities. However, basic natural history information is lacking for many terrestrial species, particularly reptiles in temperate regions of the world. To overcome this limitation, we collated 3527 reptile observations from 52 species across an endangered woodland ecoregion in south‐eastern Australia and examined ecological niche breadth and microhabitat guild structure. We found 30% of species had low ecological niche values and were classified as habitat specialists associated with large eucalypt trees, woody debris, surface rock or rocky outcrops. Cluster analysis separated species into six broad guilds based on microhabitat similarity. Approximately 80% of species belonged to guilds associated with old growth vegetation attributes or non‐renewable litho‐resources such as surface rock or rocky outcrops. Our results suggest that agri‐environment schemes that focus purely on grazing management are unlikely to provide immediate benefits to broad suites of reptiles associated with old growth vegetation and litho‐resources. Our classification scheme will be useful for identifying reptile species that are potentially vulnerable to anthropocentric disturbances and may require alternative strategies for improving habitat suitability and reptile conservation outcomes in grassy woodland ecosystems.     

Biological activity and preclinical efficacy of azetidinyl pyridazines as potent systemically-distributed stearoyl-CoA desaturase inhibitors

Potent and orally bioavailable SCD inhibitors built on an azetidinyl pyridazine scaffold were identified. In a one-month gDIO mouse model of obesity, we demonstrated that there was no therapeutic index even at low doses; efficacy in preventing weight gain tracked closely with skin and eye adverse events. This was attributed to the local SCD inhibition in these tissues as a consequence of the broad tissue distribution observed in mice for this class of compounds. The search for new structural scaffolds which may display a different tissue distribution was initiated. In preparation for an HTS campaign, a radiolabeled azetidinyl pyridazine displaying low non-specific binding in the scintillation proximity assay was prepared.     

Bicyclic heteroaryl inhibitors of stearoyl-CoA desaturase: from systemic to liver-targeting inhibitors

Optimization of a lead thiazole amide MF-152 led to the identification of potent bicyclic heteroaryl SCD1 inhibitors with good mouse pharmacokinetic profiles. In a view to target the liver for efficacy and to avoid SCD1 inhibition in the skin and eyes where adverse effects were previously observed in rodents, representative systemically-distributed SCD1 inhibitors were converted into liver-targeting SCD1 inhibitors.