A minimal model for decoding of time-limited Ca2+ oscillations

Calcium oscillations regulate several cellular processes by activating particular proteins. Most theoretical studies focused on the idealized situation of infinitely long oscillations. Here we analyze information transfer by time-limited calcium spike trains. We show that proteins can be selectively activated in a resonance-like manner by time-limited spike trains of different frequencies, while infinitely long oscillations do not show this resonance phenomenon. We found that proteins are activated more specifically by shorter oscillatory signals with narrower spikes.     

Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane

The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PF3 and PF6 observed in previous studies.     

Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines

Endosomal TLRs play an important role in innate immune response as well as in autoimmune processes. In the therapy of systemic lupus erythematosus, antimalarial drugs chloroquine, hydroxychloroquine, and quinacrine have been used for a long time. Their suppression of endosomal TLR activation has been attributed to the inhibition of endosomal acidification, which is a prerequisite for the activation of these receptors. We discovered that chloroquine inhibits only activation of endosomal TLRs by nucleic acids, whereas it augments activation of TLR8 by a small synthetic compound, R848. We detected direct binding of antimalarials to nucleic acids by spectroscopic experiments and determined their cellular colocalization. Further analysis revealed that other nucleic acid-binding compounds, such as propidium iodide, also inhibited activation of endosomal TLRs and colocalized with nucleic acids to endosomes. We found that imidazoquinolines, which are TLR7/8 agonists, inhibit TLR9 and TLR3 even in the absence of TLR7 or TLR8, and their mechanism of inhibition is similar to the antimalarials. In contrast to bafilomycin, none of the tested antimalarials and imidazoquinolines inhibited endosomal proteolysis or increased the endosomal pH, confirming that inhibition of pH acidification is not the underlying cause of inhibition. We conclude that the direct binding of inhibitors to nucleic acids mask their TLR-binding epitope and may explain the efficiency of those compounds in the treatment of autoimmune diseases.     

The cumulative effects of polymorphisms in the DNA mismatch repair genes and tobacco smoking in oesophageal cancer risk

The DNA mismatch repair (MMR) enzymes repair errors in DNA that occur during normal DNA metabolism or are induced by certain cancer-contributing exposures. We assessed the association between 10 single-nucleotide polymorphisms (SNPs) in 5 MMR genes and oesophageal cancer risk in South Africans. Prior to genotyping, SNPs were selected from the HapMap database, based on their significantly different genotypic distributions between European ancestry populations and four HapMap populations of African origin. In the Mixed Ancestry group, the MSH3 rs26279 G/G versus A/A or A/G genotype was positively associated with cancer (OR?=?2.71; 95% CI: 1.34-5.50). Similar associations were observed for PMS1 rs5742938 (GG versus AA or AG: OR?=?1.73; 95% CI: 1.07-2.79) and MLH3 rs28756991 (AA or GA versus GG: OR?=?2.07; 95% IC: 1.04-4.12). In Black individuals, however, no association between MMR polymorhisms and cancer risk was observed in individual SNP analysis. The interactions between MMR genes were evaluated using the model-based multifactor-dimensionality reduction approach, which showed a significant genetic interaction between SNPs in MSH2, MSH3 and PMS1 genes in Black and Mixed Ancestry subjects, respectively. The data also implies that pathogenesis of common polymorphisms in MMR genes is influenced by exposure to tobacco smoke. In conclusion, our findings suggest that common polymorphisms in MMR genes and/or their combined effects might be involved in the aetiology of oesophageal cancer.     

Emasculation: gloves-off strategy enhances eunuch spider endurance

Males of sexually cannibalistic spiders commonly mutilate parts of their paired genitals (palps) during copulation, which may result in complete emasculation or the 'eunuch phenomenon'. In an orb-web nephilid spider, Nephilengys malabarensis, about 75 per cent of males fall victim to sexual cannibalism, and the surviving males become half-eunuchs (one palp emasculated) or full-eunuchs (both palps emasculated). While it has been shown that surviving eunuchs are better fighters compared with intact males when guarding the females with which they have mated, mechanisms behind eunuchs' superior fighting abilities are unknown. The previously proposed 'gloves-off' hypothesis, attributing eunuchs' enhanced locomotor endurance to the reduction in total body weight caused by genital mutilation, is plausible but has remained untested. Here, we tested the gloves-off hypothesis in N. malabarensis by comparing the time until exhaustion (i.e. endurance) of intact males with half- and full-eunuchs created experimentally. We found that by reducing body weight up to 4 per cent in half-eunuchs and 9 per cent in full-eunuchs through emasculation, endurance increases significantly in half-eunuchs (32%) and particularly strongly in full-eunuchs (80%). Our results corroborate the gloves-off hypothesis and further point towards the adaptive significance of male emasculation.     

Molecular characterization of the homo-phytochelatin synthase of soybean Glycine max: relation to phytochelatin synthase.

The phytochelatin homologs homo-phytochelatins are heavy metal-binding peptides present in many legumes. To study the biosynthesis of these compounds, we have isolated and functionally expressed a cDNA GmhPCS1 encoding homo-phytochelatin synthase from Glycine max, a plant known to accumulate homo-phytochelatins rather than phytochelatins upon the exposure to heavy metals. The catalytic properties of GmhPCS1 were compared with the phytochelatin synthase AtPCS1 from Arabidopsis thaliana. When assayed only in the presence of glutathione, both enzymes catalyzed phytochelatin formation. GmhPCS1 accepted homoglutathione as the sole substrate for the synthesis of homo-phytochelatins whereas AtPCS1 did not. Homo-phytochelatin synthesis activity of both recombinant enzymes was significantly higher when glutathione was included in the reaction mixture. The incorporation of both glutathione and homoglutathione into homo-phytochelatin, n = 2, was demonstrated using GmhPCS1 and AtPCS1. In addition to bis(glutathionato)-metal complexes, various other metal-thiolates were shown to contribute to the activation of phytochelatin synthase. These complexes were not accepted as substrates by the enzyme, thereby suggesting that a recently proposed model of activation cannot fully explain the catalytic mechanism of phytochelatin synthase (Vatamaniuk, O. K., Mari, S., Lu, Y. P., and Rea, P. A. (2000) J. Biol. Chem. 275, 31451-31459).     

Zinc-decorated silica-coated magnetic nanoparticles for protein binding and controlled release

The aim of this study was to be able to reversibly bind histidine-rich proteins to the surface of maghemite magnetic nanoparticles via coordinative bonding using Zn ions as the anchoring points. We showed that in order to adsorb Zn ions on the maghemite, the surface of the latter needs to be modified. As silica is known to strongly adsorb zinc ions, we chose to modify the maghemite nanoparticles with a nanometre-thick silica layer. This layer appeared to be thin enough for the maghemite nanoparticles to preserve their superparamagnetic nature. As a model the histidine-rich protein bovine serum albumin (BSA) was used. The release of the BSA bound to Zn-decorated silica-coated maghemite nanoparticles was analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrated that the bonding of the BSA to such modified magnetic nanoparticles is highly reversible and can be controlled by an appropriate change of the external conditions, such as a pH decrease or the presence/supply of other chelating compounds.     

Controlling nucleic acid secondary structure by intercalation: effects of DNA strand length on coralyne-driven duplex disproportionation

Small molecules that intercalate in DNA and RNA are powerful agents for controlling nucleic acid structural transitions. We recently demonstrated that coralyne, a small crescent-shaped molecule, can cause the complete and irreversible disproportionation of duplex poly(dA)·poly(dT) into triplex poly(dA)·poly(dT)·poly(dT) and a poly(dA) self- structure. Both DNA secondary structures that result from duplex disproportionation are stabilized by coralyne intercalation. In the present study, we show that the kinetics and thermodynamics of coralyne-driven duplex disproportionation strongly depend on oligonucleotide length. For example, disproportionation of duplex (dA)₁₆·(dT)₁₆ by coralyne reverts over the course of hours if the sample is maintained at 4°C. Coralyne-disproportioned (dA)₃₂· (dT)₃₂, on the other hand, only partially reverts to the duplex state over the course of days at the same temperature. Furthermore, the equilibrium state of a (dA)₁₆·(dT)₁₆ sample in the presence of coralyne at room temperature contains three different secondary structures [i.e. duplex, triplex and the (dA)₁₆ self-structure]. Even the well-studied process of triplex stabilization by coralyne binding is found to be a length-dependent phenomenon and more complicated than previously appreciated. Together these observations indicate that at least one secondary structure in our nucleic acid system [i.e. duplex, triplex or (dA)_n self-structure] binds coralyne in a length-dependent manner.     

Tuning of conformational preorganization in model 2',5'- and 3',5'-linked oligonucleotides by 3'- and 2'-O-methoxyethyl modification

Conformational properties of trimeric and tetrameric 2′,5′-linked oligonucleotides, 3′-MOE-A₃^2′,5′ (1) and 3′-MOE-A₄^2′,5′ (2), and their 3′,5′-linked analogs, 2′-MOE-A₃^3′,5′ (3) and 2′-MOE-A₄^3′,5′ (4), were examined with the use of heteronuclear NMR spectroscopy. The temperature-dependent ³J_HH, ³J_HP and ³J_CP coupling constants, acquired in the range of 273–343 K, gave insight into the conformation of sugar rings in terms of a two-state North ↔ South (N ↔ S) pseudorotational equilibrium and into the conformation of the sugar–phosphate backbone in the model antisense oligonucleotides 1–4. 2′,5′-linked oligomers 3′-MOE-A₃^2′,5′ (1) and 3′-MOE-A₄^2′,5′ (2) show preference for N-type conformers and indication of A-type conformational features, which is prerequisite for antisense hybridization. The drive of N ↔ S equilibrium in 1–4 has been rationalized with the competing gauche effects of 2′/3′-phosphodiester and 3′/2′-MOE groups, anomeric and steric effects. Furthermore, the pairwise comparisons of 3′-MOE with 3′-OH and 3′-deoxy 2′,5′-linked adenine trimers emphasized the fine tuning of N ↔ S equilibrium in 3′-MOE-A₃^2′,5′ (1) and 3′-MOE-A₄^2′,5′ (2) by the steric effects of 3′-MOE group and the possibility of water-mediated H-bonds with vicinal phosphodiester functionality. In full correspondence, the drive of N ↔ S equilibrium towards N by 2′-MOE in 3′,5′-linked analogs 2′-MOE-A₃^3′,5′ (3) and 2′-MOE-A₄^3′,5′ (4) is weaker in comparison with 3′-OH group in the corresponding ribo analogs. β^t, γ⁺ and ε^– rotamers are preferred in both 2′,5′- and in 3′,5′-linked oligonucleotides 1–4.