Stacking of safranine in liposomes during valinomycininduced efflux of potassium ions

Liposomes were prepared from phosphatidylcholine and cardiolipin in a KCl medium and suspended in a choline chloride medium with safranine. When efflux of K⁺ was induced by valinomycin, spectral shifts characteristic of stacking were observed. Ca²⁺ inhibited the rate of stacking in a competitive manner with a K_i of about 200 μM, while La³⁺ was about 10 times more potent. When liposomes were prepared from phospholipids with a higher ratio of cardiolipin to phosphatidyl-choline the inhibition was more potent. No effect on the stacking phenomena was seen when Ca²⁺ was added after the stacking was completed. When Ca²⁺ or an organic cation with four charges, spermine, was trapped in the intraliposomal compartment, no significant change in the rate of stacking was seen. However, the extent of stacking was decreased. It is suggested that safranine is driven by a diffusion potential to a site that is inaccessible to Ca²⁺ in the medium, presumably to the inner boundaries of the liposomal membranes.     

The use of subunit exchange chromatography for the group specific fractionation of histones

Starting from the histone mixture obtained from calf thymus, the arginine rich fraction ARE⁺⁾ was coupled to organomercurial agarose via a mercaptide bond to one of its cysteines. ARE-agarose proved to be useful for a large scale affinity chromatographic separation of whole histone. In 1M NaCl, pH 4.5, highly pure histone fractions could be eluted with an urea gradient revealing increasing affinity towards ARE in the order: KAP < KAS < LAK < (ARE)_n(GRK)_n < GRK < ARE.     

The role of Ca-2+ and cyclic adenosine 3':5'-monophosphate in insulin release induced in vitro by the divalent cation ionophore A23187.

Insulin release from isolated perifused pancreatic islets was stimulated by the divalent ionophore A23187 in the absence of exogenous glucose. In addition, A23187 produced a 2-fold elevation of cyclic adenosine 3':5'-monophosphate (cAMP) levels in isolated perifused islets. The elevation of cAMP levels coincided with peak insulin release. Ionophore-induced insulin release was unaffected by pretreatment of the islets with theophylline (5 mM). Stimulation of insulin release produced by the ionophore occurred either in the presence or absence of extracellular Ca-2+; however, cAMP accumulation required the presence of extracellular Ca-2+. The ionophore (10 muM) had no effect on adenylate cyclase activity of homogenates of isolated islets. The results of this study are interpreted as indicating that intracellular Ca-2+ has an essential role in the insulin releasing mechanism, whereas the cAMP system has a modulatory effect on this process.     

The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers: HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN*

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE_T and BChE_T with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q_N-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q_N-GPI always consist of AChE_T and BChE_T homodimers. The dimer formation of AChE_T and BChE_T depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE_T or BChE_T homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.     

Common and rare species respond to similar niche processes in macroinvertebrate metacommunities

Ecologists have long investigated why communities are composed of a few common species and many rare species. Most studies relate rarity to either niche differentiation among species or spatial processes. There is a parallel between these processes and the processes proposed to explain the structure of metacommunities. Based on a metacommunity perspective and on data on stream macroinvertebrates from different regions of Brazil, we answer two questions. 1) Are sets of common and rare species affected by similar niche and spatial processes? 2) How does the community composition of common and of rare species differ? The main hypothesis we test is that common species are mainly affected by environmental factors, whereas rare species are mostly influenced by dispersal limitation. We used variation partitioning to determine the proportion of variation explained by the environment and space in common and rare species matrices. Contrary to our expectations, evidence supported the idea that both common and rare species are affected mainly by environmental factors, even after controlling for the differing information content between common and rare species matrices. Moreover, the abundance of some common species is also a good predictor of variation in rare species matrices. Niche differences are unlikely to be the sole cause of patterns of rarity in these metacommunities. We suggest that sets of common and rare species react to similar major environmental gradients and that rare species also respond to processes that operate at a more fine‐grained spatial scale, particularly biotic interactions. We extend the view that species sorting is the dominant process structuring metacommunities and argue that future studies focusing on rarity would benefit from a metacommunity perspective.     

Structural understanding of stabilization patterns in engineered bispecific Ig‐like antibody molecules

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

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.