Voltage sensitivities and deactivation kinetics of histamine H3 and H4 receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by inhibitory autoreceptors. Likewise, receptor deactivation rates upon agonist removal have been implicated in autoreceptor function. Using G protein-coupled potassium (GIRK) channel activation in Xenopus oocytes as readout of receptor activity, we have investigated the voltage sensitivities and signaling kinetics of the hH₃⁴⁴⁵ and hH₃³⁶⁵ isoforms of the human histamine H₃ receptor, which functions as an inhibitory auto- and heteroreceptor in the nervous system. We have also investigated both the human and the mouse homologues of the related histamine H₄ receptor, which is expressed mainly on hematopoietic cells. We found that the hH₃⁴⁴⁵ receptor is the most sensitive to voltage, whereas the hH₃³⁶⁵ and H₄ receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH₃⁴⁴⁵ and hH₃³⁶⁵ isoforms, with the hH₃³⁶⁵ isoform being five to six-fold slower than the hH₃⁴⁴⁵ receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH₄ receptor. The differences in voltage sensitivities and deactivation kinetics between the hH₃⁴⁴⁵, hH₃³⁶⁵, and H₄ receptors might be relevant to their respective physiological roles.     

Expression of the Hutchinson-Gilford Progeria Mutation during Osteoblast Development Results in Loss of Osteocytes,Irregular Mineralization,and Poor Biomechanical Properties

Hutchinson-Gilford progeria syndrome (HGPS) is a very rare genetic disorder that is characterized by multiple features of premature aging and largely affects tissues of mesenchymal origin. In this study, we describe the development of a tissue-specific mouse model that overexpresses the most common HGPS mutation (LMNA, c.1824C>T, p.G608G) in osteoblasts. Already at the age of 5 weeks, HGPS mutant mice show growth retardation, imbalanced gait and spontaneous fractures. Histopathological examination revealed an irregular bone structure, characterized by widespread loss of osteocytes, defects in mineralization, and a hypocellular red bone marrow. Computerized tomography analysis demonstrated impaired skeletal geometry and altered bone structure. The skeletal defects, which resemble the clinical features reported for bone disease in HGPS patients, was associated with an abnormal osteoblast differentiation. The osteoblast-specific expression of the HGPS mutation increased DNA damage and affected Wnt signaling. In the teeth, irregular dentin formation, as was previously demonstrated in human progeria cases, caused severe dental abnormalities affecting the incisors. The observed phenotype also shows similarities to reported bone abnormalities in aging mice and may therefore help to uncover general principles of the aging process.     

A miniaturised electrochemical affinity assay based on a wall-free sample droplet and micro-dispensing of the redox-labelled binding partner.

An affinity-assay was developed that is based on the modulation of the diffusion coefficient of a redox-labelled hapten upon complementary recognition of the analyte leading to an increase of molecular weight and hence to a decrease of the diffusion coefficient. The slower diffusion is monitored by means of cyclic voltammetry. In order to demonstrate the feasibility of this assay format, recognition of biotin by streptavidin has been chosen as a model system. Labelling of biotin was achieved by covalent binding of a ferrocene derivative to the biotin unit. To reduce the consumption of expensive compounds and to allow automatisation of the assay a novel miniaturised set-up was developed based on a wall-free sample droplet which forms the electrochemical cell with typical volumes of up to 10 microl. This droplet is dispensed by means of a step-motor driven syringe pump through a specially designed electrode holder spanning the gap between a micro-working electrode and a macroscopic counter electrode. By means of a piezo-driven micro-dispenser a predefined number of nano-droplets (100 pl volume each) containing the redox-labelled hapten are shot into the sample droplet. By this, any physical contact and hence any cross-contamination between the sample and the reagent solution could be avoided. Signal amplification can be achieved by redox recycling between the micro-electrode and the perpendicular positioned macroscopic counter electrode.     

Visualization of the eEF2-80S ribosome transition-state complex by cryo-electron microscopy

In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-Å cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the γ-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.     

Solution structure of poly(ethylene) glycol-conjugated hemoglobin revealed by small-angle X-ray scattering: implications for a new oxygen therapeutic

Developing protein therapeutics has posed challenges due to short circulating times and toxicities. Recent advances using poly(ethylene) glycol (PEG) conjugation have improved their performance. A PEG-conjugated hemoglobin (Hb), Hemospan, is in clinical trials as an oxygen therapeutic. Solutions of PEG-hemoglobin with two (P5K2) or six to seven strands of 5-kD PEG (P5K6) were studied by small-angle x-ray scattering. PEGylation elongates the dimensions (Hb < P5K2 < P5K6) and leaves the tertiary hemoglobin structure unchanged but compacts its quaternary structure. The major part of the PEG chains visualized by ab initio reconstruction protrudes away from hemoglobin, whereas the rest interacts with the protein. PEGylation introduces intermolecular repulsion, increasing with conjugated PEG amount. These results demonstrate how PEG surface shielding and intermolecular repulsion may prolong intravascular retention and lack of reactivity of PEG-Hb, possibly by inhibiting binding to the macrophage CD163 hemoglobin-scavenger receptor. The proposed methodology for assessment of low-resolution structures and interactions is a powerful means for rational design of PEGylated therapeutic agents.     

TaqMan-Based Real-Time PCR Method for Detection of Yersinia pseudotuberculosis in Food

A sensitive and specific assay for detection of food-borne pathogenic Yersinia pseudotuberculosis was developed. The primer-probe set was designed to target a 157-bp sequence of the chromosomally located gene ail. The complete method, including an internal amplification control, was evaluated for several different food items.     

Molecular correlates for maximal oxygen uptake and type 1 fibers

Maximal oxygen uptake (Vo(2max)) and the amount of type 1 fibers are interrelated, but the underlying unifying molecular mechanisms are poorly understood. To explore these mechanisms, we related gene expression profiles in skeletal muscle biopsies of 43 age-matched men from published datasets with Vo(2max) and the amount of type 1 fibers and replicated some of the findings in muscle biopsies from 154 young and elderly individuals using real-time PCR. We identified 66 probe sets (genes or expressed sequence tags) positively and 83 probe sets inversely correlated with Vo(2max) and 171 probe sets positively and 217 probe sets inversely correlated with percentage of type 1 fibers in human skeletal muscle. Genes involved in oxidative phosphorylation (OXPHOS) showed high expression in individuals with high Vo(2max), whereas the opposite was not the case in individuals with low Vo(2max). Instead, genes such as AHNAK and BCL6 were associated with low Vo(2max). Also, expression of the OXPHOS genes NDUFB5 and ATP5C1 increased with exercise training and decreased with aging. In contrast, expression of AHNAK in skeletal muscle decreased with exercise training and increased with aging. Eleven genes (NDUFB4, COX5A, UQCRB, ATP5C1, ATP5G3, ETHE1, FABP3, ISCA1, MYST4, C9orf3, and PKIA) were positively correlated with both Vo(2max) and the percentage of type 1 fibers. Vo(2max) closely reflects expression of OXPHOS genes, particularly that of NDUFB5 and ATP5C1, in skeletal muscle, suggesting good muscle fitness. In contrast, a high expression of AHNAK was associated with a low Vo(2max) and poor muscle fitness.     

Aminomethyl-2,6-difluorophenols as a novel class of increased lipophilicity GABA(C) receptor antagonists

3- and 4-(Aminomethyl)-2,6-difuorophenols were tested for activity against the three major classes of GABA receptors. 4-(Amninomethyl)-2,6difluorophenol was shown to be a competitive and somewhat selective antagonist at p1 GABA(C) receptors expressed in Xenopus oocytes (K(B) = 75.5 microM with a 95% Confidence Interval range of 75.2 microM to 75.8 microM). This is the first in a novel class of increased lipophilicity GABA(C) receptor antagonists with little activity at alpha1beta2gamma2 GABA(A) and GABA(B) receptors.     

Aboveground and belowground responses to quality and heterogeneity of organic inputs to the boreal forest

Leaf litter and other organic resources returned to the soil are important regulators of ecological processes in forest ecosystems, and their ecological impacts may be strongly influenced both by their quality and by interactions between coexisting resource types. To date, most studies on effects of resource identity and mixing have only involved leaf litter, despite the fact that other resource types constitute a major input to the soil. We investigated how quality and heterogeneity of organic substrates found in boreal forests affects the activity and community structure of soil microbes, and plant growth. Six organic substrates (wood, charcoal, berries, sporocarps, vertebrate faeces and leaf litter) were added singly or in mixtures of two, three and six resource types to pots containing forest soil (with or without tree seedlings of Betula pendula Roth). The largest positive effects of single substrates on microbial basal respiration (BR), substrate-induced respiration (SIR) and microbial metabolic quotient (qCO₂) were found for nutrient-rich substrates (faeces and sporocarps) or substrates with high sugar-content (berries). Mixing of substrates had no effect on BR or SIR, but decreased qCO₂ or altered the microbial community structure for specific combinations of substrates. In contrast to the niche complementarity hypothesis, microbial catabolic diversity was not stimulated by greater diversity of resources. Seedling growth responses to single substrates were neutral or negative; the inhibition of growth probably resulted largely from microbial competition for nutrients. Substrate mixing enhanced seedling nutrient-uptake and growth for all mixtures containing sporocarps and leaf litter. Overall, plants responded more strongly to resource heterogeneity than microbes, and synergistic effects only occurred when nutrient-rich substrates were present within the substrate mixtures. In particular, our results demonstrate a role for complex and non-additive interactions among previously overlooked resource types returned to the soil in influencing ecosystem functions such as nutrient cycling and plant productivity.