Complexation of siRNA with Dendrimer: A Molecular Modeling Approach

This paper reports the structural behavior and thermodynamics of the complexation of siRNA with poly(amidoamine) (PAMAM) dendrimers of generation 3 (G3) and 4 (G4) through fully atomistic molecular dynamics (MD) simulations accompanied by free energy calculations and inherent structure determination. We have also done simulation with one siRNA and two dendrimers (2 x G3 or 2xG4) to get the microscopic picture of various binding modes. Our simulation results reveal the formation of stable siRNA-dendrimer complex over nanosecond time scale. With the increase in dendrimcr generation, the charge ratio increases and hence the binding energy between siRNA and dendrimer also increases in accordance with available experimental measurements. Calculated radial distribution functions of amines groups of various subgenerations in a given generation of dendrimer and phosphate in backbone of siRNA reveals that one dendrimer of generation 4 shows better binding with siRNA almost wrapping the dendrimer when compared to the binding with lower generation dendrimer like G3. In contrast, two dendrimers of generation 4 show binding without siRNA wrapping the den-rimer because of repulsion between two dendrimers. The counterion distribution around the complex and the water molecules in the hydration shell of siRNA give microscopic picture of the binding dynamics. We see a clear correlation between water. counterions motions and the complexation i.e. the water molecules and counterions which condensed around siRNA are moved away from the siRNA backbone when dendrimer start binding to the siRNA back hone. As siRNA wraps/bind to the dendrimer counterions originally condensed onto siRNA (Na-1) and dendrimer (Cl-) get released. We give a quantitative estimate of the entropy of counterions and show that there is gain in entropy due to counterions release during the complexation. Furthermore, the free energy of complexation of IG3 and IG4 at two different salt concentrations shows that increase in salt concentration leads to the weakening of the binding affinity of siRNA and dendrimer.

Modelling the chemical weathering fluxes at the watershed scale in the Tropics (Mule Hole, South India): Relative contribution of the smectite/kaolinite assemblage versus primary minerals

We investigate the chemical weathering processes and fluxes in a small experimental watershed (SEW) through a modelling approach. The study site is the Mule Hole SEW developed on a gneissic basement located in the climatic gradient of the Western Ghats, South India. The model couples a lumped hydrological model simulating the water budget at the watershed scale to the WITCH model estimating the dissolution/precipitation rates of minerals using laboratory kinetic laws. Forcing functions and parameters of the simulation are defined by the field data. The coupled model is calibrated with stream and groundwater compositions through the testing of a large range of smectite solubility and abundance in the soil horizons. We found that, despite the low abundance of smectite in the dominant soil type of the watershed (4 vol.%), their net dissolution provides 75% of the export of dissolved silica, while primary silicate mineral dissolution releases only 15% of this flux. Overall, smectites (modelled as montmorillonites) are not stable under the present day climatic conditions. Furthermore, the dissolution of trace carbonates in the saprolitic horizon provides 50% of the calcium export at the watershed scale. Modelling results show the contrasted behavior of the two main soil types of the watershed: red soils (88% of the surface) are provider of calcium, while black soils (smectite-rich and characterized by a lower drainage) consumes calcium through overall carbonate precipitation. Our model results stress the key role played by minor/accessory minerals and by the thermodynamic properties of smectite minerals, and by the drainage of the weathering profiles on the weathering budget of a tropical watershed. (C) 2010 Elsevier B.V. All rights reserved.

Protein Kinase C and Anaplastic Lymphoma Kinase Targeted Compounds

The protein kinases (PKs) belong to the largest single family of enzymes, phosphotransferases, which catalyze the phosphorylation of other enzymes and proteins and function primarily in signal transduction. Consequently, PKs regulate cell mechanisms such as growth, differentiation, and proliferation. Dysfunction of these cellular mechanisms may lead to cancer, a major predicament in health care. Even though there is a range of clinically available cancer-fighting drugs, increasing number of cancer cases and setbacks such as drug resistance, constantly keep cancer research active. At the commencement of this study an isophthalic acid derivative had been suggested to bind to the regulatory domain of protein kinase C (PKC). In order to investigate the biological effects and structure-activity relationships (SARs) of this new chemical entity, a library of compounds was synthesized. The best compounds induced apoptosis in human leukemia HL-60 cells and were not cytotoxic in Swiss 3T3 fibroblasts. In addition, the best apoptosis inducers were neither cytotoxic nor mutagenic. Furthermore, results from binding affinity assays of PKC isoforms revealed the pharmacophores of these isophthalic acid derivatives. The best inhibition constants of the tested compounds were measured to 210 nM for PKCα and to 530 nM for PKCδ. Among natural compounds targeting the regulatory domain of PKC, the target of bistramide A has been a matter of debate. It was initially found to activate PKCδ; however, actin was recently reported as the main target. In order to clarify and to further study the biological effects of bistramide A, the total syntheses of the natural compound and two isomers were performed. Biological assays of the compounds revealed accumulation of 4n polyploid cells as the primary mode of action and the compounds showed similar overall antiproliferative activities. However, each compound showed a distinct distribution of antimitotic effect presumably via actin binding, proapoptotic effect presumably via PKCδ, and pro-differentiation effect as evidenced by CD11b expression. Furthermore, it was shown that the antimitotic and proapoptotic effects of bistramide A were not secondary effects of actin binding but independent effects. The third aim in this study was to synthesize a library of a new class of urea-based type II inhibitors targeted at the kinase domain of anaplastic lymphoma kinase (ALK). The best compounds in this library showed IC50 values as low as 390 nM for ALK while the initial low cellular activities were successfully increased even by more than 70 times for NPM-ALK- positive BaF3 cells. More importantly, selective antiproliferative activity on ALK-positive cell lines was achieved; while the best compound affected the BaF3 and SU-DHL-1 cells with IC50 values of 0.5 and 0.8 μM, respectively, they were less toxic to the NPM-ALK-negative human leukemic cells U937 (IC50 = 3.2 μM) and BaF3 parental cells (IC50 = 5.4 μM). Furthermore, SAR studies of the synthesized compounds revealed functional groups and positions of the scaffold, which enhanced the enzymatic and cellular activities.

Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia

2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 μImage for 2,4-dichlorophenol, and 100 μImage for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.

Large low-frequency resistance noise in chemical vapor deposited graphene

We report a detailed investigation of resistance noise in single layer graphene films on Si/SiO2 substrates obtained by chemical vapor deposition (CVD) on copper foils. We find that noise in these systems to be rather large, and when expressed in the form of phenomenological Hooge equation, it corresponds to Hooge parameter as large as 0.1-0.5. We also find the variation in the noise magnitude with the gate voltage (or carrier density) and temperature to be surprisingly weak, which is also unlike the behavior of noise in other forms of graphene, in particular those from exfoliation. (C) 2010 American Institute of Physics. doi:10.1063/1.3493655]

Influence of cross-correlation between dipolar and chemical shift anisotropy relaxation mechanisms upon the transverse relaxation rates of 15N in macromolecules

Heteronuclear multiple-quantum coherence relaxation rate are calculated for the individual transitions of the S spin in an AIS nuclear spin system assuming that the heteronucleus (S spin) has relaxation contributions from both intramolecular dipole-dipole and chemical shift anisotropy relaxation. The individual multiplet components of the heteronuclear zero- and double-quantum coherences are shown to have different transverse relaxation rates. The cross-correlation between the two relaxation mechanisms is shown to be the dominant cause of the calculated differential line broadening. Experimental data are presented using as an example a uniformly 15N labelled sample of human epidermal growth factor.

Mechanistic evaluation of mitigation of petroleum hydrocarbon contamination by soil medium

Present in situ chemical treatment technologies for mitigation of petroleum hydrocarbon contamination are in the developmental stage or being tested. To devise efficient strategies for restricting the movement of petroleum hydrocarbon (PHC) molecules in the contaminated soil, it is proposed to utilize the sorption–interaction relationships between the petroleum contaminants and the soil substrate. The basic questions addressed in this paper are as follows (i) What are the prominent chemical constituents of the various petroleum fractions that interact with the soil substrate? (ii) What are the functional groups of a soil that interact with the contaminants? (iii) What are the bonding mechanisms possible between the soil functional groups and the PHC contaminants? (iv) What are the consequent changes brought about the soil physical properties on interaction with PHC's? (v) What are the factors influencing the interactions between PHC molecules and clay particles of the soil substrate? (vi) What is the possibility of improving the soil's attenuation ability for PHC's? The development of answers to the basic questions reveal that petroleum hydrocarbons comprise a mixture of nonpolar alkanes and aromatic and polycyclic hydrocarbons, that have limited solubility in water. The bonding mechanism between the nonpolar PHC's and the clay surface is by way of van der Waals attraction. The adsorption of the nonpolar hydrocarbons by the clay surface occurs only when their (i.e., the hydrocarbon molecules) solubility in water is exceeded and the hydrocarbons exist in the micellar form. Dilute solutions of hydrocarbons in water, i.e., concentrations of hydrocarbons at or below the solubility limit, have no effect on the hydraulic conductivity of clay soils. Permeation with pure hydrocarbons invariably influences the clay hydraulic conductivity. To improve the attenuation ability of soils towards PHC's, it is proposed to coat the soil surface with "ultra" heavy organic polymers. Adsorption of organic polymers by the clay surface may change the surface properties of the soil from highly hydrophilic (having affinity for water molecules) to organophilic (having affinity for organic molecules). The organic polymers attached to the clay surface are expected to attenuate the PHC molecules by van der Waals attraction, by hydrogen bonding, and also by adsorption into interlayer space in the case of soils containing swelling clays.

Purification and characterization of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-Sepharose affinity chromatography

Three toxins, abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. abrin-I did not bind on DEAE-Sephacel column chromatography and the bound abrin-II, abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are abrin-I, 64,000; abrin-II and abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms−1 at a 0.63 nImage concentration of each of abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic abrin-I and the highly toxic abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.

Relation between effective atomic charge and chemical shifts in X-ray absorption spectra of transition-metal compounds

Chemical shifts of K absorption discontinuities, Delta E, of several manganese, iron and cobalt oxides with the metal in the formal oxidation states between +2 and +4, have been measured. These data, together with data in the literature on other compounds of these metals, can be fitted into the expression Delta E=aq+bq2, where q is the effective atomic charge on the metal. Theoretical considerations also support this functional relationship between Delta E and q.

29Si NMR chemical shifts in silicates

29Si chemical shifts in a wide variety of silicates in crystalline, glassy and gel states have been related to a parameter, P, which takes into account the electronegativity and the structural description of the silicate units as well as the ionic potential of the modifier cation. The relation, δ(ppm)=28.4 [1−exp(−P)]−110.5, besides having predictive value, satisfactorily accounts for all the available chemical-shifts data on silicates and shows the right kind of limiting behaviour, with δ approaching the Q0 value at large P.

Plant aspartate transcarbamylase: An affinity chromatographic method for the purification of the enzyme from germinated seedlings

A simple and rapid affinity chromatographic method for the isolation of aspartate transcarbamylase from germinated seedlings of mung bean (Phaseolus aureus) was developed. A partially purified preparation of the enzyme was chromatographed on an affinity column containing aspartate linked to CNBr-activated Sepharose 4B. Aspartate transcarbamylase was specifically eluted from the column with 10 mImage aspartate or 0.5 Image KCl. The enzyme migrated as a single sharp band during disc electrophoresis at pH 8.6 on polyacrylamide gels. Electrophoresis of the sodium dodecyl sulfate-treated enzyme showed two distinct protein bands, suggesting that the mung bean aspartate transcarbamylase was made up of nonidentical subunits. Like the enzyme purified by conventional procedures, this enzyme preparation also exhibited positive homotropic interactions with carbamyl phosphate and negative heterotropic interactions with UMP. This method was extended to the purification of aspartate transcarbamylase from Lathyrus sativus, Eleucine coracona, and Trigonella foenum graecum.