Specific binding of Ro 09-0198 (cinnamycin) to phosphatidylethanolamine: a thermodynamic analysis

Ro 09-0198 (cinnamycin) is a tetracyclic peptide antibiotic that is used to monitor the transbilayer movement of phosphatidylethanolamine (PE) in biological membranes during cell division and apoptosis. The molecule is one of the very rare examples where a small peptide binds specifically to a particular lipid. In model membranes and biological membranes containing phosphatidylethanolamine, Ro 09-0198 forms a 1:1 complex with this lipid. We have measured the thermodynamic parameters of complex formation with high sensitivity isothermal titration calorimetry and have investigated the structural consequences with deuterium and phosphorus solid-state NMR. Complex formation is characterized by a large binding constant, K0, of 10(7) to 10(8) M(-1), depending on the experimental conditions. The reaction enthalpy, DeltaHdegrees, varies between zero at 10 degrees C to strongly exothermic -10 kcal/mol at 50 degrees C. For large vesicles with a diameter of approximately 100 nm, DeltaHdegrees decreases linearly with temperature and the molar heat capacity of complex formation can be evaluated as = -245 cal/mol, indicating a hydrophobic binding mechanism. The free energy of binding is DeltaGdegrees = -10.5 kcal/mol and shows only little temperature dependence. The constancy of DeltaGdegrees together with the distinct temperature-dependence of DeltaHdegrees provide evidence for an entropy-enthalpy compensation mechanism: at 10 degrees C, complex formation is completely entropy-driven, at 50 degrees C it is enthalpy-driven. Varying the PE fatty acid chain-length between 6 and 18 carbon atoms produces similar binding constants and DeltaHdegrees values. Addition of Ro 09-0198 to PE containing bilayers eliminates the typical bilayer structure and produces 2H- and 31P-NMR spectra characteristic of slow isotropic tumbling. This reorganization of the lipid matrix is not limited to PE but also includes other lipids.     

Quantitative determination of nuclear pore complexes in cycling cells with differing DNA content

The number of pore complexes per nucleus was determined for a wide variety of cultured cells selected for their variable DNA content over a range of 1-5,6000. The pore number was compared to DNA content, nuclear surface area, and nuclear volume. Values for pore frequency (pores/square micrometer) were relatively constant in the species studied. When the pore to DNA ratio was plotted against the DNA content, there was a remarkable correlation which decreased exponentially for the cells of vertebrae origin. Exceptions were the heteroploid mammalian cells which had the same ratio as the diploid mammalian cells despite higher DNA content. The results are interpreted to mean that neither the nuclear surface, the nuclear volume, nor the DNA content alone determines the pore number of the nucleus, but rather an as yet undetermined combination of different factors. The surface and volume of vertebrate nuclei do not decrease with decreasing DNA content below a given value. The following speculation is suggested to account for the anomalous size changes of the nucleus relative to DNA content in vertebrates. Species with small DNA complements have a relatively large proportion of active chromatin which determines the limits of the physical parameters of the nucleus. The amount of active chromatin maybe the same for at least the vertebrates with low DNA content, At high DNA content, the nuclear parameters may be determined by the relatively high proportion of inactive condensed chromatin which increases the nuclear surface and volume.     

Inhibition of phosphodiesterase 4 modulates cytokine induction from toll like receptor activated,but not rhinovirus infected,primary human airway smooth muscle

Background

Virus-induced exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are a significant health burden and occur even in those receiving the best current therapies. Rhinovirus (RV) infections are responsible for half of all COPD exacerbations. The mechanism by which exacerbations occur remains undefined, however it is likely to be due to virus-induced inflammation. Given that phophodiesterase 4 (PDE₄) inhibitors have an anti-inflammatory effect in patients with COPD they present a potential therapy prior to, and during, these exacerbations.

Methods

In the present study we investigated whether the PDE₄ inhibitor piclamilast (10^-6 M) could alter RV or viral mimetic (5 μg/mL of imiquimod or poly I:C) induced inflammation and RV replication in primary human airway smooth muscle cells (ASMC) and bronchial epithelial cells (HBEC). The mediators IL-6, IL-8, prostaglandin E₂ and cAMP production were assayed by ELISA and RV replication was assayed by viral titration.

Results

We found that in ASMCs the TLR3 agonist poly I:C induced IL-8 release was reduced while induced IL-6 release by the TLR7/8 agonist imiquimod was further increased by the presence of piclamilast. However, in RV infected ASMCs, virus replication and induced mediator release were unaltered by piclamilast, as was also found in HBECs. The novel findings of this study reveal that although PDE inhibitors may not influence RV-induced cytokine production in ASMCs and replication in either ASMCs or HBECs, they have the capacity to be anti-inflammatory during TLR activation by modulating the induction of these chemotactic cytokines.

Conclusion

By extrapolating our in vitro findings to exacerbations of COPD in vivo this suggests that PDE₄ inhibitors may have beneficial anti-inflammatory properties when patients are infected with bacteria or viruses other than RV.     

Nine new polymorphic microsatellite loci for the amplification of archived otolith DNA of Atlantic cod,Gadus morhua L.

Nine out of 22 microsatellite primers tested were successfully amplified on three samples of cod Gadus morhua L. (two contemporary and one archived otolith samples). All loci were polymorphic (5–23 alleles/locus). The average observed heterozygosity across loci and samples was 0.625, ranging from 0.294 to 0.895 at each locus. All loci were under Hardy–Weinberg equilibrium, except PGmo56 that showed significant excess of heterozygotes in all studied samples. The isolated loci were suitable for degraded DNA and therefore useful for conducting a long‐term temporal study with DNA obtained from archived otoliths of cod.     

Mangafodipir a Selective Cytoprotectant — with Special Reference to Oxaliplatin and Its Association to Chemotherapy-Induced Peripheral Neuropathy (CIPN)

Oxaliplatin, in combination with 5-fluorouracil plus folinate (or capecitabine), has increased survival substantially in stage III colorectal cancer and prolonged life in stage IV patients, but its use is compromised because of severe toxicity. Chemotherapy-induced peripheral neuropathy (CIPN) is the most problematic dose-limiting toxicity of oxaliplatin. Oncologists included for years calcium and magnesium infusion as part of clinical practice for preventing CIPN. Results from a phase III prospective study published in 2014, however, overturned this practice. No other treatments have been clinically proven to prevent this toxicity. There is a body of evidence that CIPN is caused by cellular oxidative stress. Clinical and preclinical data suggest that the manganese chelate and superoxide dismutase mimetic mangafodipir (MnDPDP) is an efficacious inhibitor of CIPN and other conditions caused by cellular oxidative stress, without interfering negatively with the tumoricidal activity of chemotherapy. MnPLED, the metabolite of MnDPDP, attacks cellular oxidative stress at several critical levels. Firstly, MnPLED catalyzes dismutation of superoxide (O₂^??), and secondly, having a tremendous high affinity for iron (and copper), PLED binds and disarms redox active iron/copper, which is involved in several detrimental oxidative steps. A case report from 2009 and a recent feasibility study suggest that MnDPDP may prevent or even cure oxaliplatin-induced CIPN. Preliminary results from a phase II study (PLIANT) suggest efficacy also of calmangafodipir, but these results are according to available data obscured by a surprisingly low number of adverse events and a seemingly lower than expected efficacy of FOLFOX.     

Mechanisms of Xenogeneic Baboon Platelet Aggregation and Phagocytosis by Porcine Liver Sinusoidal Endothelial Cells

Background

Baboons receiving xenogeneic livers from wild type and transgenic pigs survive less than 10 days. One of the major issues is the early development of profound thrombocytopenia that results in fatal hemorrhage. Histological examination of xenotransplanted livers has shown baboon platelet activation, phagocytosis and sequestration within the sinusoids. In order to study the mechanisms of platelet consumption in liver xenotransplantation, we have developed an in vitro system to examine the interaction between pig endothelial cells with baboon platelets and to thereby identify molecular mechanisms and therapies.

Methods

Fresh pig hepatocytes, liver sinusoidal and aortic endothelial cells were isolated by collagenase digestion of livers and processing of aortae from GTKO and Gal+ MGH-miniature swine. These primary cell cultures were then tested for the differential ability to induce baboon or pig platelet aggregation. Phagocytosis was evaluated by direct observation of CFSE labeled-platelets, which are incubated with endothelial cells under confocal light microscopy. Aurintricarboxylic acid (GpIb antagonist blocking interactions with von Willebrand factor/vWF), eptifibatide (Gp IIb/IIIa antagonist), and anti-Mac-1 Ab (anti-α_Mβ₂ integrin Ab) were tested for the ability to inhibit phagocytosis.

Results

None of the pig cells induced aggregation or phagocytosis of porcine platelets. However, pig hepatocytes, liver sinusoidal and aortic endothelial cells (GTKO and Gal+) all induced moderate aggregation of baboon platelets. Importantly, pig liver sinusoidal endothelial cells efficiently phagocytosed baboon platelets, while pig aortic endothelial cells and hepatocytes had minimal effects on platelet numbers. Anti-MAC-1 Ab, aurintricarboxylic acid or eptifibatide, significantly decreased baboon platelet phagocytosis by pig liver endothelial cells (P<0.01).

Conclusions

Although pig hepatocytes and aortic endothelial cells directly caused aggregation of baboon platelets, only pig liver endothelial cells efficiently phagocytosed baboon platelets. Blocking vWF and integrin adhesion pathways prevented both aggregation and phagocytosis.     

Polarity of microtubules nucleated by centrosomes and chromosomes of Chinese hamster ovary cells in vitro

The structural and growth polarities of centrosomal and chromosomal microtubules were studied by analyzing the kinetics of growth of these microtubules and those initiated by flagellar seeds. By comparing rates of elongation of centrosomal and flagellar-seeded microtubules, we determined whether the centrosomal microtubules were free to grow at their plus ends only, minus ends ony, or at both ends. Our results show that centrosomal microtubules elongate at a rate corresponding to the addition of subunits at the plus end only. The depolymerization rate was also equivalent to that for the plus end only. Chromosomal microtubule elongation was similar to the centrosome-initiated growth. Since the data do not support the hypothesis that both ends of these spindle microtubules are able to interact with monomer in solution, then growth must occur only distal or only proximal to the organizing centers, implying tha the opposite ends in unavailable for exchange of subunits. Experiments with flagellar-seeded microtubules serving as internal controls indicated that the inactivity of the minus end could not be accounted for by a diffusible inhibitor, suggesting a structural explanation. Since there is no apparent way in which the distal ends may be capped, whereas the proximal ends are embedded in the pericentriolar cloud, we conclude that centrosomal microtubules are oriented with their plus ends distal to the site of nucleation. A similar analysis for chromosomal microtubules suggests that they too must be oriented with their plus ends distal to the site of initiation.     

Recolonization history and large-scale dispersal in the open sea: the case study of the North Atlantic cod, Gadus morhua L.

Most studies of the genetic structure of Atlantic cod have focused on small geographical scales. In the present study, the genetic structure of cod sampled on spawning grounds in the North Atlantic was examined using eight microsatellite loci and the Pan I locus. A total of 954 cod was collected from nine different regions: the Baltic Sea, the North Sea, the Celtic Sea, the Irish Sea and Icelandic waters during spring 2002 and spring 2003, from Norwegian waters and the Faroe Islands (North and West spawning grounds) in spring 2003, and from Canadian waters in 1998. Temporal stability among spawning grounds was observed in Icelandic waters and the Celtic Sea, and no significant difference was observed between the samples from the Baltic Sea and between the samples from Faroese waters. F -statistics showed significant differences between most populations and a pattern of isolation-by-distance was described with microsatellite loci. The Pan I locus revealed the presence of two genetically distinguishable basins, the North-west Atlantic composed of the Icelandic and Canadian samples and the North-east Atlantic composed of all other samples. Permutation of allele sizes at each microsatellite locus among allelic states supported a mutational component to the genetic differentiation, indicating a historical origin of the observed variation. Estimation of the time of divergence was approximately 3000 generations, which places the origin of current genetic pattern of cod in the North Atlantic in the late Weichselian (Wisconsinian period), at last glacial maximum.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 315–329.     

Actin Filament Bundling and Different Nucleating Effects of Mouse Diaphanous-Related Formin FH2 Domains on Actin/ADF and Actin/Cofilin Complexes

Mouse Diaphanous-related formins (mDias) are members of the formin protein family that nucleate actin polymerization and subsequently promote filamentous actin (F-actin) elongation by monomer addition to fast-growing barbed ends. It has been suggested that mDias preferentially recruit actin complexed to profilin due to their proline-rich FH1 domains. During filament elongation, dimeric mDias remain attached to the barbed ends by their FH2 domains, which form an anti-parallel ring-like structure enclosing the filament barbed ends. Dimer formation of mDia-FH2 domains is dependent on their N-terminal lasso and linker subdomains (connector). Here, we investigated the effect of isolated FH2 domains on actin polymerization using mDia1-FH2 domain plus connector, as well as core mDia1, mDia2, and mDia3 missing the connector, by cosedimentation and electron microscopy after negative staining. Analytical ultracentrifugation showed that core FH2 domains of mDia1 and mDia2 exhibited a low degree of dimer formation, whereas mDia3-FH2 minus connector and mDia1-FH2 plus connector readily dimerized. Only core mDia3-FH2 was able to nucleate actin polymerization. However, all tested core FH2 domains decorated and bundled F-actin, as demonstrated by electron microscopy after negative staining. Bundling activity was highest for mDia3-FH2, decreased for mDia2-FH2, and further decreased for mDia1-FH2. The mDia1-FH2 domain plus connector induced actin polymerization also in the absence of profilin, but failed to induce F-actin deformation and bundling. We also tested whether mDia1-FH2 was able to repolymerize actin in complex with different proteins that stabilize globular actin. The data obtained demonstrated that mDia1-FH2 induced actin repolymerization only from the actin/cofilin-1 complex, but not when complexed to actin depolymerizing factor, gelsolin segment 1, vitamin D binding protein, or deoxyribonuclease I.     

Adhesion and clustering of charge isomers of myelin basic protein at model myelin membranes

The association of myelin basic protein charge isomers with the lipid part of the myelin membrane was investigated at the microscopic (molecular) level in a model membrane system, using optical waveguide lightmode spectrometry to determine with high precision the kinetics of association and dissociation to planar phospholipid membranes under controlled hydrodynamic conditions and over a range of protein concentrations. Detailed analysis of the data revealed a rich and intricate behaviour and clearly showed that the membrane protein affinity is characterized by at least four independent parameters: (i) the association rate coefficient characterizing the protein-membrane interaction energy as the protein approaches the fluid-membrane interface; (ii) the protein-membrane adhesion, i.e., the probability that it will remain at the membrane after arrival; (iii) the protein conformation at the membrane; and (iv) the protein's tendency to cluster at the membrane. Some of these parameters varied in characteristic ways as the bulk solution concentration of the protein was varied, giving further clues to the detailed molecular comportment of the protein. The parameters and their characteristic variations with bulk concentration were markedly different for the different isomers. Implications of these results for neurological disorders involving demyelination, such as multiple sclerosis, are discussed.