Structural changes in dipalmitoylphosphatidylcholine bilayer promoted by Ca2+ ions: a small-angle neutron scattering study

Small-angle neutron scattering (SANS) curves of unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles in 1-60mM CaCl2 were analyzed using a strip-function model of the phospholipid bilayer. The fraction of Ca2+ ions bound in the DPPC polar head group region was determined using Langmuir adsorption isotherm. In the gel phase, at 20 degrees C, the lipid bilayer thickness, dL, goes through a maximum as a function of CaCl2 concentration (dL=54.4A at approximately 2.5mM of CaCl2). Simultaneously, both the area per DPPC molecule AL, and the number of water molecules nW located in the polar head group region decrease (DeltaAL=AL(DPPC))-AL(DPPC+Ca)=2.3A2 and Deltan=n(W(DPPC))-n(W(DPPC+Ca))=0.8mol/mol at approximately 2.5mM of CaCl2). In the fluid phase, at 60 degrees C, the structural parameters d(L), A(L), and n(W) show evident changes with increasing Ca2+ up to a concentration C(Ca)(2+) < or = 10mM. DPPC bilayers affected by the calcium binding are compared to unilamellar vesicles prepared by extrusion. The structural parameters of DPPC vesicles prepared in 60mM CaCl2 (at 20 and 60 degrees C) are nearly the same as those for unilamellar vesicles without Ca2+.     

Synthesis, DNA interaction, and cytotoxic activity of a novel proflavine-dithiazolidinone pharmacophore

Five novel proflavine-dithiazolidinone derivatives 4a-4e have been designed and synthesized by the reaction of dialkyl acridin-3,6-diyl dithioureas 3a-3e with methyl bromoacetate. The binding affinity of dithiazolidinone hydrochlorides 5a-5e with calf thymus DNA and plasmid (pUC19) DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence, and CD spectroscopy. The effects of 5a-5e on the thermal denaturation profiles of calf thymus DNA were also studied. From spectrophotometric and spectrofluorimetric titrations, the binding constants for the pUC19 DNA-drug complexes were determined (K = 6.2-2.2 x 104 M-1). In vitro cytotoxic activities of compounds 5a-5e toward murine leukemia cell line L1210 and human uterus carcinoma HeLa cells were also examined. 2',2' '-[(Acridin-3,6-diyl)diimino]-3',3' '-dipropyl-1,3-dithiazolidin-4-one hydrochloride (5b) showed the highest activity against these cells with IC50 values of 6.3 microM and 12.9 microM over the course of 72 h.     

Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction

BACKGROUND: Shot (previously named Kakapo), is a Drosophila Plakin family member containing both Actin binding and microtubule binding domains. In Drosophila, it is required for a wide range of processes, including axon extension, dendrite formation, axonal terminal arborization at the neuromuscular junction, tendon cell development, and adhesion of wing epithelium. RESULTS: To address how Shot exerts its activity at the molecular level, we investigated the molecular interactions of Shot with candidate proteins in mature larval tendon cells. We show that Shot colocalizes with EB1/APC1 and with a compact microtubule array extending between the muscle-tendon junction and the cuticle. Shot forms a protein complex with EB1 via its C-terminal EF-hands and GAS2-containing domains. In tendon cells with reduced Shot activity, EB1/APC1 dissociate from the muscle-tendon junction, and the microtubule array elongates. The resulting tendon cell, although associated with the muscle and the cuticle ends, loses its stress resistance and elongates. CONCLUSIONS: Our results suggest that Shot mediates tendon stress resistance by the organization of a compact microtubule network at the muscle-tendon junction. This is achieved by Shot association with the cytoplasmic faces of the basal hemiadherens junction and with the EB1/APC1 complex.     

Asymptotics and bioavailability in a 17-compartment pharmacokinetic model with enterohepatic circulation and remetabolization

A 17-compartment linear pharmacokinetic model is designed, describing the complex process of enterohepatic circulation as a superposition of the net (remetabolizationfree) enterohepatic circulation, and remetabolization with subsequent intestinal absorption of the parent drug. Basically, the model is built by doubling the model describing the circulation of the parent drug in the body, so that the remetabolizable metabolite circulates in a model of the same structure as does the parent compound. The two submodels are cross-connected with arrows denoting the transition of the particular substance into the complementary part of the complex model. Asymptotic properties of the model are investigated, in particular, explicit formulas for its pharmacokinetic endpoints are given using the elements of its transition probability matrix. Conversely, taking account of the effect of bile cannulation, intravenous, intraportal and oral administration of the drug, as well as of the intravenous and intraportal administration of the remetabolizable metabolite, the transition probabilities of the system are determined in terms of certain measurable pharmacokinetic endpoints and the flow rates through the kidneys, liver and the cardiac output. Finally, the influence of the enterohepatic circulation and remetabolization process on bioavailability is examined. In particular, the inclusion-exclusion formula is derived, expressing its joint efficiency (defined as the relative increase of bioavailability) by means of the efficiencies of the net enterohepatic circulation and of the remetabolization process.     

Exploratory Search on Twitter Utilizing User Feedback and Multi-Perspective Microblog Analysis

In recent years, besides typical information retrieval, a broader concept of information exploration – exploratory search - is emerging into the foreground. In addition, more and more valuable information is presented in microblogs on social networks. We propose a new method for supporting the exploratory search on the Twitter social network. The method copes with several challenges, namely brevity of microblogs called tweets, limited number of available ratings and the need to process the recommendations online. In order to tackle the first challenge, the representation of microblogs is enriched by information from referenced links, topic summarization and affect analysis. The small number of available ratings is raised by interpreting implicit feedback trained by feedback model during browsing. Recommendations are made by a preference model that models user’s preferences over tweets. The evaluation shows promising results even when navigating in the space of brief pieces of information, making recommendations based only on a small number of ratings, and by optimizing the models to process in real time.     

Hypericin Site Specific Interactions Within Polynucleotides Used as DNA Model Compounds

Summary

Interactions of the antiretroviral hypericin molecule with polynucleotides, i.e. poly(dG-dC), poly(dA-dT), poly(rG) and poly(rC), have been studied in aqueous solutions by resonance Raman spectroscopy, using an UV excitation wavelength which induces a specific resonance enhancement of spectral band intensities corresponding to proper nucleic base modes of vibration. It is shown that : i) hypericin selectively interacts with the N7 sites of purines, ii) the strength of interaction depends on the polymer structure, and : iii) interaction with guanine is stronger than with adenine molecules.     

Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris

Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZalphaA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 +/- 3.7 micromol/min/mg of protein with 1,2-dibromoethane and 1.86 +/- 0.36 micromol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7+/-0.3 degrees C for LinB expressed in P. pastoris and 41.8 +/- 0.3 degrees C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.     

Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.