Diethylstilbestrol Modifies the Structure of Model Membranes and Is Localized Close to the First Carbons of the Fatty Acyl Chains.

The synthetic estrogen diethylstilbestrol (DES) is used to treat metastatic carcinomas and prostate cancer. We studied its interaction with membranes and its localization to understand its mechanism of action and side-effects. We used differential scanning calorimetry (DSC) showing that DES fluidized the membrane and has poor solubility in DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) in the fluid state. Using small-angle X-ray diffraction (SAXD), it was observed that DES increased the thickness of the water layer between phospholipid membranes, indicating effects on the membrane surface. DSC, X-ray diffraction, and 31P-NMR spectroscopy were used to study the effect of DES on the Lα-to-HII phase transition, and it was observed that negative curvature of the membrane is promoted by DES, and this effect may be significant to understand its action on membrane enzymes. Using the 1H-NOESY-NMR-MAS technique, cross-relaxation rates for different protons of DES with POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) protons were calculated, suggesting that the most likely location of DES in the membrane is with the main axis parallel to the surface and close to the first carbons of the fatty acyl chains of POPC. Molecular dynamics simulations were in close agreements with the experimental results regarding the location of DES in phospholipids bilayers.

Autoradiography and Phosphorimaging.

Many of the commonly used techniques in molecular cloning depend on methods to map accurately the distribution of radioactive atoms on two-dimensional (2D) surfaces. Without this ability, methods such as Southern blotting, northern hybridizations, radiolabeled DNA sequencing, and library screening would not have been possible. In the 1970s and 1980s-the pioneering days of molecular cloning-imaging of 2D surfaces was obtained using autoradiography. In this technique, ß-particles emitted by radioactive specimens were recorded on X-ray film, producing a latent image that can be converted to a true image by developing and fixing the film. Autoradiography was a lot of fun, but it was also messy. In the impatient excitement of wanting to see how an experiment had turned out, people used to hold the newly developed X-ray films in their metal frames up to the darkroom light. Drips of the final wash would run down their arms, clothes would be stained, and shoes ruined. It is hardly surprising that autoradiography was quickly abandoned when sensitive phosphorimagers came onto the market at the end of the 1990s.

Time-Dependent Lipid Dynamics, Organization and Peptide-Lipid Interaction in Phospholipid Bilayers with Incorporated ß-Amyloid Oligomers.

Nonfibrillar ß-amyloid (Aß) oligomers are considered as major neurotoxic species in the pathology of Alzheimer's disease. The presence of Aß oligomers was shown to cause membrane disruptions in a broad range of model systems. However, the molecular basis of such a disruption process remains unknown. We previously demonstrated that membrane-incorporated 40-residue Aß (Aß40) oligomers could form coaggregates with phospholipids. This process occurred more rapidly than the fibrillization of Aß40 and led to more severe membrane disruption. The present study probes the time-dependent changes in lipid dynamics, bilayer structures, and peptide-lipid interactions along the time course of the oligomer-induced membrane disruption, using solid-state NMR spectroscopy. Our results suggest the presence of certain intermediate states with phospholipid molecules entering the C-terminal hydrogen-bonding networks of the Aß40 oligomeric cores. This work provides insights on the molecular mechanisms of Aß40-oligomer-induced membrane disruption.

Rhein-PEG-nHA conjugate as a bone targeted drug delivery vehicle for enhanced cancer chemoradiotherapy.

Bone-targeted therapies have been the choice of treatments for cancer metastases in bone to minimize skeletal morbidity and preserve patients' quality of life. Rhein is of particular interest due to its high bone affinity. Here we reported a novel Rhein- polyethylene glycol (PEG)-nano hydroxyapatite (nHA) conjugate to deliver doxorubicin (DOX) and Phosphorus-32 (32P) simultaneously for enhanced cancer chemo-radiotherapy. The synthetic Rhein-PEG-nHA conjugates were sphere in shape with an average diameter of ~120 nm. Their morphology, drug release and bone affinity were confirmed in vitro. The release profiles of DOX depend on pH condition, but 32P exhibited good stability. Rhein-PEG-nHA also showed high bone affinity in vivo, and the tumor volume decreased after the DOX@Rhein-PEG-nHA and 32P@Rhein-PEG-nHA treatments. Most importantly, the DOX/32P@Rhein-PEG-nHA showed the strongest inhibition on the growth of bone metastases of breast cancer. We revealed the potential of Rhein-PEG-nHA in combined chemo-radiation treatment for bone metastases of breast cancer.

31P and 13C solid-state NMR analysis of morphological changes of phospholipid bilayers containing glucagon during fibril formation of glucagon under neutral condition.

Glucagon is a 29 amino acid peptide hormone secreted by pancreatic α-cells that interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibrillar aggregates that are cytotoxic due to their activation of apoptotic signaling pathways. To understand the glucagon-membrane interactions, morphological changes in dimyristoylphosphatidylcholine (DMPC) bilayers containing glucagon in neutral solution were investigated by observing 31P NMR spectra. First, lipid bilayers with a DMPC/glucagon molar ratio of 50/1 were observed. One day after preparing the DMPC/glucagon lipid bilayer sample, lipid bilayers were disrupted below the phase transition temperature (Tc). Membrane disruption was reduced 2 days after preparation due to the reduction of glucagon-DMPC interaction, and subsequently increased by 4 days and was reduced again by 7 days. TEM measurements showed that small ellipsoidal intermediates of glucagon were observed inside the small size of lipid bilayer after 4 days, while fibrils grew inside lipid bilayer after 19 days. These results indicate that morphological changes in DMPC/glucagon lipid bilayers are correlated with the evolution of glucagon aggregate state. Particularly, fibril intermediate shows a strong glucagon lipid bilayer interaction. We further investigated the structure and kinetics of glucagon fibril formation inside the DMPC lipid bilayer in a neutral solution using 13C solid-state NMR spectroscopy. α-Helical structures were observed around Gly4 and Ala19 in the monomeric form, which changed to ß-sheet structures in the fibril form. The fibrillation process can be explained by a two-step autocatalytic reaction mechanism in which the first step is a homogeneous nuclear formation (k1), and the second step is an autocatalytic heterogeneous fibrillation process (k2).

Regulation and Control of AP-1 Binding Activity in Embryotoxicity.

The electrophoretic mobility shift assay (EMSA) is a sensitive and relatively straightforward methodology used to detect sequence-specific DNA-protein interactions. It is the fundamental procedure of several variants that allow qualitative and quantitative assessments of protein-nucleic acid complexes. Classically, nuclear proteins and DNA are combined, and the resulting mixture is electrophoretically separated in polyacrylamide or agarose gel under native conditions. The distribution within the gel is generally detected with autoradiography of the 32P-labelled DNA. The underlying principle is that nucleic acid with protein bound to it will migrate more slowly through a gel matrix than the free nucleic acid. In this chapter, a representative protocol is described that addresses specific challenges of using whole embryos as the nuclear protein source, and the most common and informative EMSA variant, the "super-shift", is also presented. The important points are underscored, and approaches for troubleshooting are explained. References are provided for alternative methods and extensions of the basic protocol.

Gel-Based Assays for Measuring DNA Unwinding, Annealing, and Strand Exchange.

Efficient replication and repair of the genome requires a multitude of protein-DNA transactions. These interactions can result in a variety of consequences for DNA such as the unwinding of double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), the annealing of complementary ssDNAs, or the exchange of ssDNA with one strand of a dsDNA duplex. Some DNA helicases possess all three activities, but many DNA-interacting proteins can also catalyze one or more of these reactions. Assays that quantify these activities are an important first step in characterizing these protein-DNA interactions in vitro. Here, we describe methods for the formation of dsDNA substrates and the assays that can be used to biochemically characterize proteins that can unwind, anneal, and/or exchange DNA strands.

Highlighting protein fining residues in a model red wine.

Many studies have dealt with fining treatments, focusing on their impact on phenolic precipitation or sensory properties of the treated wines. Previous articles suggested the presence of soluble complexes with tannins and fining proteins, and probably wine polysaccharides too. However, no study has quantified these possible protein residues in wine. The analyses performed on a model red wine to highlight the residual fining proteins were the measurement of viscosity, the quantification of amino acid/proteins of the samples and the radioactivity of the supernatants obtained after fining with a radioactive protein. This work has clearly shown, both qualitatively and quantitatively, the presence of fining residues in a model red wine that had been fined with a gelatin and two hydrolyzed plant proteins. These results have significant implications, yet to be confirmed with commercial fining agents and 'real' wines for allergen residues in treated wines.

Measuring the Activities of Two-Component Regulatory System Phosphatases.

Two-component regulatory systems (TCSs) are used for signal transduction by organisms from all three phylogenetic domains of the living world. TCSs use transient protein phosphorylation and dephosphorylation reactions to convert stimuli into appropriate responses to changing environmental conditions. Phosphoryl groups flow from ATP to sensor kinases (which detect stimuli) to response regulators (which implement responses) to inorganic phosphate (Pi). The phosphorylation state of response regulators controls their output activity. The rate at which phosphoryl groups are removed from response regulators correlates with the timescale of the corresponding biological function. Dephosphorylation reactions are fastest in chemotaxis TCS and slower in other TCS. Response regulators catalyze their own dephosphorylation, but at least five types of phosphatases are known to enhance dephosphorylation of response regulators. In each case, the phosphatases are believed to stimulate the intrinsic autodephosphorylation reaction. We discuss in depth the properties of TCS (particularly the differences between chemotaxis and nonchemotaxis TCS) relevant to designing in vitro assays for TCS phosphatases. We describe detailed assay methods for chemotaxis TCS phosphatases using loss of 32P, change in intrinsic fluorescence as a result of dephosphorylation, or release of Pi. The phosphatase activities of nonchemotaxis TCS phosphatases are less well characterized. We consider how the properties of nonchemotaxis TCS affect assay design and suggest suitable modifications for phosphatases from nonchemotaxis TCS, with an emphasis on the Pi release method.

Evaluation of Bremsstrahlung radiation dose in stereotactically radiocolloid therapy of cystic craniopharyngioma tumors with 32P radio-colloid.

Over 90% of craniopharyngeal brain tumors are cystic, which enables the injection of beta emitters such as phosphorus-32 (32P) radio-colloid into cysts for their treatment. The aim of this study was to evaluate the clinical and theoretical modelling of Bremsstrahlung radiation dose resulting from stereotactic radio-colloid therapy of cystic craniopharyngioma tumors with 32P. 32P radio-colloid with appropriate activity concentration was injected to a head phantom, and then the Bremsstrahlung radiation spectrum and planar images were obtained using a gamma camera. Both phantom and gamma camera were simulated using MCNPX code, and the results were compared with practical results. Bremsstrahlung radiation spectrum was measured using a handheld gamma spectrometer for two patients treated with stereotactic radio-colloid therapy with 32P in different positions and compared to Monte Carlo simulation. Results of counting and determining sensitivity coefficients in the air and the attenuating environment were obtained. Also, comparing the counting sensitivity from practical and simulation methods indicated the agreement of the data between the two methods. Comparison of the spectra from different positions around patient's head indicated the ability to use this detector to quantify the activity in the operating room. Selection of the spectrum is important in Bremsstrahlung radiation imaging. We can take advantage of spectrometry measurement using gamma camera, handheld gamma spectrometer for patient, and theoretical modeling with Monte Carlo code to evaluate radiopharmaceutical distribution, leakage, as well as estimate activity and predict therapeutic effects in other adjacent structures and ultimately optimize radio-colloid therapy in cystic craniopharyngeal patients.

Reconstituting the 4-Strand DNA Strand Exchange.

Proteins of the Rad51 family play a key role in homologous recombination by carrying out DNA strand exchange. Here, we present the methodology and the protocols for the 4-strand exchange between gapped circular DNA and homologous linear duplex DNA promoted by human Rad51 and Escherichia coli RecA orthologs. This reaction includes formation of joint molecules and their extension by branch migration in a polar manner. The presented methodology may be used for reconstitution of the medial-to-late stages of homologous recombination in vitro as well as for investigation of the mechanisms of branch migration by helicase-like proteins, e.g., Rad54, BLM, or RecQ1.

GEN1 Endonuclease: Purification and Nuclease Assays.

Successful chromosome segregation depends on the timely removal of DNA recombination and replication intermediates that interlink sister chromatids. These intermediates are acted upon by structure-selective endonucleases that promote incisions close to the junction point. GEN1, a member of the Rad2/XPG endonuclease family, was identified on the basis of its ability to cleave Holliday junction recombination intermediates. Resolution occurs by a nick and counter-nick mechanism in strands that are symmetrically related across the junction point, leading to the formation of ligatable nicked duplex products. The actions of GEN1 are, however, not restricted to HJs, as 5'-flaps and replication fork structures also serve as excellent in vitro substrates for the nuclease. In the cellular context, GEN1 activity is observed late in the cell cycle, as most of the protein is excluded from the nucleus, such that it gains access to DNA intermediates after the breakdown of nuclear envelope. Nuclear exclusion ensures the protection of replication forks and other DNA secondary structures important for normal metabolic processes. In this chapter, we describe the purification of recombinant GEN1 and detail biochemical assays involving the use of synthetic DNA substrates and cruciform-containing plasmids.

Preparation and Resolution of Holliday Junction DNA Recombination Intermediates.

Holliday junctions provide a covalent link between recombining DNA molecules and need to be removed prior to chromosome segregation at mitosis. Defects in their resolution lead to mitotic catastrophe, characterized by the formation of DNA breaks and chromosome aberrations. Enzymes that resolve recombination intermediates have been identified in all forms of life, from bacteriophage, to bacteria, yeast, and humans. In higher eukaryotes, Holliday junctions are resolved by GEN1, a nuclease that is mechanistically similar to the prototypic resolvase Escherichia coli RuvC, and by the SMX trinuclease complex. Studies of these enzymes have been facilitated by the use of plasmid-sized DNA recombination intermediates made by RecA-mediated strand exchange. Here, we detail the preparation of these recombination intermediates, which resemble α-structures, and their resolution by RuvC and GEN1.

Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK.

AMP-activated protein kinase (AMPK) is an energy sensor that is activated by increases in the cellular AMP/ATP and ADP/ATP ratios by three mechanisms: (1) allosteric activation, (2) promotion of phosphorylation at Thr172 on the α subunit by upstream kinases, and (3) inhibition of dephosphorylation of Thr172 by protein phosphatases. All of these effects are triggered by the binding of AMP or ADP at one or more of three sites on the γ subunit, where they displace ATP. AMPK is also activated by ligands that bind in the ADaM site, which is located between the α and ß subunits. In this chapter we describe cell-free assays that can be used to study these varied activation mechanisms.

Bioinformatics Approach to Identify Novel AMPK Targets.

In silico analysis of Big Data is a useful tool to identify putative kinase targets as well as nodes of signaling cascades that are difficult to discover by traditional single molecule experimentation. System approaches that use a multi-tiered investigational methodology have been instrumental in advancing our understanding of cellular mechanisms that result in phenotypic changes. Here, we present a bioinformatics approach to identify AMP-activated protein kinase (AMPK) target proteins on a proteome-wide scale and an in vitro method for preliminary validation of these targets. This approach offers an initial screening for the identification of AMPK targets that can be further validated using mutagenesis and molecular biology techniques.

Synthesis of [32P]-c-di-GMP for Diguanylate Cyclase and Phosphodiesterase Activity Determinations.

Diguanylate cyclases that synthesize and phosphodiesterases that hydrolyze the second messenger cyclic-di-GMP (c-di-GMP) are at the center of bacterial signaling pathways that control behaviors relevant to all aspects of microbial physiology and pathogenesis (Romling et al., Microbiol Mol Biol Rev 77(1):1-52, 2013). Bioinformatics tools can easily predict the presence of the diguanylate cyclase GGDEF domain, or the EAL and HD-GYP domains associated with phosphodiesterase activity. However, experimental confirmation of enzymatic activity is still necessary, as many proteins contain degenerate domains that lack catalytic activity but nonetheless function as c-di-GMP receptors.

Determining Phosphodiesterase Activity (Radioactive Assay).

Cyclic-di-GMP phosphodiesterases (PDEs) catalyze the hydrolysis of the bacterial second messenger c-di-GMP. This protocol describes a sensitive radioactive assay for PDE activity in which substrate and product can be quickly and easily separated by thin-layer chromatography.

SPOt: A novel and streamlined microarray platform for observing cellular tRNA levels.

Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. It is modular and easily streamlined with popular techniques such as polysome fractionation to profile tRNAs interacting with ribosomes and actively engaged in translation. The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome.

Evaluating the Application of Tissue-Specific Dose Kernels Instead of Water Dose Kernels in Internal Dosimetry: A Monte Carlo Study.

PURPOSE: The aim of this work is to evaluate the application of tissue-specific dose kernels instead of water dose kernels to improve the accuracy of patient-specific dosimetry by taking tissue heterogeneities into consideration. MATERIALS AND METHODS: Tissue-specific dose point kernels (DPKs) and dose voxel kernels (DVKs) for yttrium-90 (90Y), lutetium-177 (177Lu), and phosphorus-32 (32P) are calculated using the Monte Carlo (MC) simulation code GATE (version 7). The calculated DPKs for bone, lung, adipose, breast, heart, intestine, kidney, liver, and spleen are compared with those of water. The dose distribution in normal and tumorous tissues in lung, liver, and bone of a Zubal phantom is calculated using tissue-specific DVKs instead of those of water in conventional methods. For a tumor defined in a heterogeneous region in the Zubal phantom, the absorbed dose is calculated using a proposed algorithm, taking tissue heterogeneity into account. The algorithm is validated against full MC simulations. RESULTS: The simulation results indicate that the highest differences between water and other tissue DPKs occur in bone for 90Y (12.2% ± 0.6%), 32P (18.8% ± 1.3%), and 177Lu (16.9% ± 1.3%). The second highest discrepancy corresponds to the lung for 90Y (6.3% ± 0.2%), 32P (8.9% ± 0.4%), and 177Lu (7.7% ± 0.3%). For 90Y, the mean absorbed dose in tumorous and normal tissues is calculated using tissue-specific DVKs in lung, liver, and bone. The results are compared with doses calculated considering the Zubal phantom water equivalent and the relative differences are 4.50%, 0.73%, and 12.23%, respectively. For the tumor in the heterogeneous region of the Zubal phantom that includes lung, liver, and bone, the relative difference between mean calculated dose in tumorous and normal tissues based on the proposed algorithm and the values obtained from full MC dosimetry is 5.18%. CONCLUSIONS: A novel technique is proposed considering tissue-specific dose kernels in the dose calculation algorithm. This algorithm potentially enables patient-specific dosimetry and improves estimation of the average absorbed dose of 90Y in a tumor located in lung, bone, and soft tissue interface by 6.98% compared with the conventional methods.

Hydroxyapatite (HA) microparticles labeled with (32)P - A promising option in the radiation synovectomy for inflamed joints.

In the present article we describe a systematic approach pursued for the synthesis of (32)P-labeled hydroxyapatite (HA) microparticles (1-10µm size range) using no carrier added (NCA) (32)P produced in a nuclear reactor and animal evaluation of its utility as an expected viable radiopharmaceutical for the treatment of pain intensive arthrosis. NCA (32)P was produced via the (32)S(n,p)(32)P route in nuclear reactor with high radionuclidic purity (99.95±0.01%, n=5). Phosphorus-32-labeled hydroxyapatite microparticles (1-10µm size range) were synthesized with high radiochemical purity (99.0±0.3% n=12) under optimized conditions and the formulation showed excellent in vitro stability in saline as well as in rat serum. Intra-articular administration of the radiolabeled particles in the knee joints of normal Wistar rats showed near-complete retention of activity within the synovial cavity upto 1 month post-administration. The radiochemical formulation thus demonstrated promising features as a radiopharmaceutical for treatment of arthritis with excellent logistic advantage for shipment to sites distant from the production facility thanks to the suitable nuclear decay properties of (32)P.