Inhibition of cGMP-dependent protein kinase II by its own splice isoform

cGMP- and cAMP-dependent protein kinases (cGK I, cGK II, and cAK) are important mediators of many signaling pathways that increase cyclic nucleotide concentrations and ultimately phosphorylation of substrates vital to cellular functions. Here we demonstrate a novel mRNA splice isoform of cGK II arising from alternative 5' splicing within exon 11. The novel splice variant encodes a protein (cGK II Delta(441-469)) lacking 29 amino acids of the cGK II Mg-ATP-binding/catalytic domain, including the conserved glycine-rich loop consensus motif Gly-x-Gly-x-x-Gly-x-Val which interacts with ATP in the protein kinase family of enzymes. cGK II Delta(441-469) has no intrinsic enzymatic activity itself, however, it antagonizes cGK II and cGK I, but not cAK. Thus, the activation and cellular functions of cGK II may be determined not only by intracellular cGMP levels but also by alternative splicing which may regulate the balance of expression of cGK II versus its own inhibitor, cGK II Delta(441-469).     

P2-P1' macrocyclization of P2 phenylglycine based HCV NS3 protease inhibitors using ring-closing metathesis

Macrocyclization is a commonly used strategy to preorganize HCV NS3 protease inhibitors in their bioactive conformation. Moreover, macrocyclization generally leads to greater stability and improved pharmacokinetic properties. In HCV NS3 protease inhibitors, it has been shown to be beneficial to include a vinylated phenylglycine in the P2 position in combination with alkenylic P1' substituents. A series of 14-, 15- and 16-membered macrocyclic HCV NS3 protease inhibitors with the linker connecting the P2 phenylglycine and the alkenylic P1' were synthesized by ring-closing metathesis, using both microwave and conventional heating. Besides formation of the expected macrocycles in cis and trans configuration as major products, both ring-contracted and double-bond migrated isomers were obtained, in particular during formation of the smaller rings (14- and 15-membered rings). All inhibitors had K(i)-values in the nanomolar range, but only one inhibitor type was improved by rigidification. The loss in inhibitory effect can be attributed to a disruption of the beneficial π-π interaction between the P2 fragment and H57, which proved to be especially deleterious for the d-phenylglycine epimers.     

Methanol and other VOC fluxes from a Danish beech forest during late springtime

In-canopy mixing ratio gradients and above-canopy fluxes of several volatile organic compounds (VOCs) were measured using a commercial proton transfer reaction mass spectrometer (PTR-MS) in a European beech (Fagus sylvatica) forest in Denmark. Fluxes of methanol were bidirectional: Emission occurred during both day and night with highest fluxes (0.2 mg C m⁻² h⁻¹) during a warm period; deposition occurred dominantly at daytime. Confirming previous branch-level measurements on beech, the forest’s monoterpene emissions (0–0.5 mg C m⁻² h⁻¹), and in-canopy mixing ratios showed a diurnal cycle consistent with light-dependent emissions; a result contrasting temperature-only driven emissions of most conifer species. Also emitted was acetone, but only at ambient temperatures exceeding 20°C. Slow deposition dominated at lower temperatures. Our in-canopy gradient measurements contrast with earlier results from tropical and pine forest ecosystems in that they did not show this beech ecosystem to be a strong sink for oxygenated VOCs (OVOCs). Instead, their gradients were flat and only small deposition velocities (<0.2 cm s⁻¹) were observed to the onsite soil. However, as methanol soil uptake was consistent and possibly related to soil moisture, more measurements are needed to evaluate its soil sink strength. In turn, as canopy scale fluxes are net fluxes with stomatal emissions from photosynthesizing leaves potentially affecting non-stomatal oxygenated VOC uptake, only independent, controlled laboratory experiments may be successful in separating gross fluxes.     

Monocomponent endoglucanase treatment increases the reactivity of softwood sulphite dissolving pulp

Softwood dissolving pulp was treated with a commercial monocomponent fungal endocellulase. The reactivity of the pulp for the production of rayon and cellulose derivatives as determined with the Fock method increased drastically with relatively low amounts of enzyme, and the yield loss and decrease of viscosity were moderate. The mechanism behind the increased reactivity is discussed.     

[3H]Imipramine Binding of Protein Nature in Human Platelets: Inhibition by 5-Hydroxytryptamine and 5-Hydroxytryptamine Uptake Inhibitors

Abstract: The nature of [³H]imipramine binding to human platelets was investigated. Desipramine and 5-hydroxytryptamine (5-HT) displaced the same amount of binding and the binding was sensitive to protease treatment. The nature of pharmacological inhibition of [³H]imipramine binding was investigated in saturation experiments. Increases in K d without changes in B _max were noted with the addition of 5-HT, desipramine, norzimeldine, or 5-methoxytryptoline. Reductions in B _max without alterations in K _D were obtained when citalopram or clomipramine was added. It is concluded that the [³H]imipramine binding site in human platelets is of protein nature and that this binding site contains the substrate recognition site for 5-HT uptake. In addition, [³H]imipramine and other 5-HT uptake inhibitors have bonds to other parts of the 5-HT uptake carrier or to the surrounding lipid membrane. This additional binding outside the substrate recognition site is not one single site but most likely represents sites that are specific for the chemical structure of each uptake inhibitor, respectively.     

Characterization of the primary radical pair in reaction centers of Heliobacillus mobilis by 13C photo-CIDNP MAS NMR

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in membrane fragments of heliobacterium Heliobacillus mobilis without further isolation by (13)C magic-angle spinning (MAS) solid-state NMR under continuous illumination with white light. In the (13)C photo-CIDNP MAS NMR spectra of heliobacterial membrane fragments, two sets of signals are observed, allowing characterization of the primary radical pair. One set, showing enhanced absorptive (positive) signals, arises from the BChl g donor, while the set of emissive (negative) signals is assigned to the 8(1)-hydroxy Chl a acceptor. Hence, under these sample conditions, both donor and acceptor sides are either monomeric or composed of identical cofactors. The occurrence of the differential relaxation (DR) mechanism suggests a donor triplet lifetime in the microsecond range. It appears that the occurrence of the solid-state photo-CIDNP effect is a general feature of primary radical pairs in natural photosynthesis.     

Implications for molecular mechanisms of glycoprotein hormone receptors using a new sequence-structure-function analysis resource

Comparison between wild-type and mutated glycoprotein hormone receptors (GPHRs), TSH receptor, FSH receptor, and LH-chorionic gonadotropin receptor is established to identify determinants involved in molecular activation mechanism. The basic aims of the current work are 1) the discrimination of receptor phenotypes according to the differences between activity states they represent, 2) the assignment of classified phenotypes to three-dimensional structural positions to reveal 3) functional-structural hot spots and 4) interrelations between determinants that are responsible for corresponding activity states. Because it is hard to survey the vast amount of pathogenic and site-directed mutations at GPHRs and to improve an almost isolated consideration of individual point mutations, we present a system for systematic and diversified sequence-structure-function analysis (http://www.fmp-berlin.de/ssfa). To combine all mutagenesis data into one set, we converted the functional data into unified scaled values. This at least enables their comparison in a rough classification manner. In this study we describe the compiled data set and a wide spectrum of functions for user-driven searches and classification of receptor functionalities such as cell surface expression, maximum of hormone binding capability, and basal as well as hormone-induced Galphas/Galphaq mediated cAMP/inositol phosphate accumulation. Complementary to known databases, our data set and bioinformatics tools allow functional and biochemical specificities to be linked with spatial features to reveal concealed structure-function relationships by a semiquantitative analysis. A comprehensive discrimination of specificities of pathogenic mutations and in vitro mutant phenotypes and their relation to signaling mechanisms of GPHRs demonstrates the utility of sequence-structure-function analysis. Moreover, new interrelations of determinants important for selective G protein-mediated activation of GPHRs are resumed.     

Faunal composition of geometrid moths changes with altitude in an Andean montane rain forest

Aim The objective of this study was to describe and interpret the changes in faunal composition in the moth family Geometridae (Lepidoptera) along a small‐scale elevational gradient in a tropical montane rain forest. This gradient was compared with a large‐scale latitudinal gradient in Europe. Location Investigations were carried out in the province Zamora‐Chinchipe in southern Ecuador along a gradient ranging from 1040 to 2677 m above sea level at twenty‐two sites. Methods Moths were sampled with light‐traps in three field periods in 1999 and 2000 and subsequently sorted and determined to species or morphospecies. Results We analysed 13,938 specimens representing 1010 species of geometrid moths. The proportional contribution of subtaxa to the local geometrid fauna changes along the elevational gradient at all systematic levels considered. While proportions of species of the subfamilies Ennominae, Sterrhinae and Geometrinae significantly decrease, the proportion of Larentiinae increases with increasing altitude. Changes also occur within the subfamilies Ennominae and Larentiinae. The host–plant specialist ennomine tribes Cassymini, Macariini and Palyadini completely vanish, and the proportion of the tribe Boarmiini decreases at high altitudes. In contrast, the remaining tribes (mostly comprising polyphagous species) either do not show proportional changes (Azelinini, Nacophorini, Nephodiini, Ourapterygini) or even increase (Caberini, ‘Cratoptera group’). Within Larentiinae, the species proportion of the genus Eois decreases, whereas concomitantly the proportion of Eupithecia increases. There is a remarkable similarity between the altitudinal patterns in Ecuador and those found along the latitudinal gradient in Europe. Main conclusions Species of the subfamily Larentiinae seem to be particularly well‐adapted to harsh environmental conditions, towards both high altitudes and latitudes. They might disproportionately profit from lower predation at higher altitudes. Many changes in the faunal composition can be explained by expected host–plant requirements of the species involved. Our results show that diversity estimates based on taxon ratios which are assumed to be constant must be regarded with caution because such ratios can change rapidly along environmental gradients.     

In situ generation of iminodiacetic acid groups on nanoporous alumina for the reversible immobilization of enzymes and other biomolecules

Nanoporous alumina membranes were silanized with aminopropylsilane and iminodiacetic acid (IDA) groups were generated in situ by reaction with iodoacetate. The membranes were mounted in standard filter holders, connected to a HPLC system and saturated with selected metal ions. Cu(II) allowed the capture of chicken muscle lactate dehydrogenase with such stability, repeatability and reproducibility that Michaelis–Menten kinetics could be studied. The IDA surface was stable for months and could be depleted and regenerated with metal ions multiple times without appreciable loss of capacity. The binding of lactate dehydrogenase influenced the backpressure to the extent that could be expected for a monolayer according to Poiseuilles law.