Monitoring liposome-mediated delivery and fate of an antisense drug in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used for evaluation of the effectiveness of delivery and fate of a model 25-mer DNA-based phosphorothiate antisense drug in cells. The antisense molecule was delivered to the cells through a simple incubation and by using a cationic liposome (Cytofectin GS 3815). The cationic lipid interacted with the negatively charged antisense to form a more positively charged complex. It was observed that uptake of the liposome-antisense complex by the cell was dependent on concentration of lipid, duration of transfection, and the cell type. The antisense drug interacted with intracellular components such as proteins and additional steps were needed to quantify the free antisense. Proteinase-K was able to release antisense from proteins. However, the addition of sodium dodecyl sulfate (SDS) to the sample or running buffer was more effective than Proteinase-K to release both naked and liposome-bound antisense from the cellular materials. Analysis of single HeLa cells for uptake of the unbound and liposome-complexed antisense revealed averages of 8.9x10(-19) moles and 4.9x10(-18) moles, respectively. The amount of uptake, however, varied greatly among individual cells and depended on the delivery method. With liposome-mediated delivery, the relative standard deviation (RSD) for the amount of antisense in individual cells was 130%, while the variation was much smaller (RSD = 45%) when the cells were incubated with the unbound antisense. These uptake variations agreed with those obtained from flow cytometry analysis.     

Crystal structures of 1-(2-hydroxyphenyl)-3-phenyl-1,3-propanedione and 1-(1,3-benzodioxol-5-yl)-3-(2,4-dimethoxyphenyl)-1,3-propanedione

1-(2-Hydroxyphenyl)-3-phenyl-1,3-propanedione crystallizes in the triclinic space group (a=5.4233(5),b=13.910(1),c=17.036(1) Å, =68.311(6), =80.854(7), =78.760(8)°) as two independent enolic tautomers in which the hydroxyl and phenolic protons are hydrogen bonded to the ketonic oxygen atom. The structure was refined toR=0.039 for 2085I3(I) reflections. 1-(1,3-Benzodioxol-5-yl)-3-(2,4-dimethoxyphenyl)-1,3-propanedione, which belongs to the triclinic space group (a=7.3990(7),b=8.1239(5),c=14.004(1) Å, =86.673(6). =88.574(7), =64.885(7)°) also exists in the enolic form. The structure was refined toR=0.040 for 1564I3(I) reflections.     

Dynamic 1H NMR Study of Aryl-Nitrogen Single Bond and Carbon-Carbon Double Bond Rotational Energy Barriers in Two Highly Functionalized Pyranopyrimidines

Summary.  The reactive 1:1 intermediate produced in the reaction between 2,6-dimethylphenyl isocyanide and dimethyl acetylenedicarboxylate was trapped by N,N′ -dimethylbarbituric acid to yield the isomeric products dimethyl 7-(2,6-dimethylphenylamino)-1,3-dimethyl-2,4-dioxo-4H-pyrano[3,2-d]pyrimidine-5,6-dicarboxylate and dimethyl (E)-2-((2,6-dimethylphenylamino)-(1,3-dimethyl-2,4,6-trioxo-pyrimidine-5-ylidene)-methyl)-but-2-enedioate in a nearly 1:1 ratio and an overall yield of 85%. Dynamic effects were observed in the ¹H NMR spectra of these compounds and were attributed to restricted rotation around the aryl-nitrogen single bonds and the polarized carbon-carbon double bond. Received September 18, 2000. Accepted (revised) November 22, 2000     

Application of internal standardization to rapid coprecipitation technique using lanthanum phosphate for flame atomic absorption spectrometric determination of iron and lead.

By applying an internal standardization, we could use a rapid coprecipitation technique using lanthanum phosphate as a coprecipitant for preconcentration of iron(III) and lead in their flame atomic absorption spectrometric determination. Indium as an internal standard was added to the initial sample solution together with lanthanum and phosphoric acid; the coprecipitation of iron(III) and lead was then carried out at pH about 3. After measuring the atomic absorbances of iron, lead, and indium in the final sample solution, we determined the contents of iron(III) and lead in the original sample solution by using the internal standardization with indium. In this method, complete collection of the precipitate was not required after the coprecipitation of iron(III), lead, and indium, because the ratio of the recovery of iron(III) or lead to that of indium was almost constant regardless of the recovery of the precipitate. This method was simple and rapid, and was available for the determination of 2-300 micrograms L-1 of iron(III) and 5-400 micrograms L-1 of lead in some water samples.     

Transition metal NMR chemical shifts and polarizability effect in organometallic complexes

The literature data on substituent influence on the ⁵¹V, ⁵⁵Mn, ⁵⁷Fe, ⁵⁹Co, ⁶¹Ni, ⁹⁵Mo, ¹⁰³Rh, ¹⁸³W, ¹⁸⁷Os and ¹⁹⁵Pt NMR chemical shifts (δ) and on J (M, P; M = Mn, Fe, Mo, Rh, W, Os) coupling constants have been analyzed for 30 series of the organometallic complexes. It has been established for the first time that the δ and J values depend on the inductive, resonance and polarizability effects of substituents. The polarizability effect is caused by the partial charge on the central M atom. The contribution of this effect ranges from 3 to 86%. Copyright © 2009 John Wiley & Sons, Ltd.     

Search for supernova relic neutrinos at Super-Kamiokande

A search for the relic neutrinos from all past core-collapse supernovae was conducted using 1496 days of data from the Super-Kamiokande detector. This analysis looked for electron-type antineutrinos that had produced a positron with an energy greater than 18 MeV. In the absence of a signal, 90% C.L. upper limits on the total flux were set for several theoretical models; these limits ranged from 20 to 130 macro nu(e) cm(-2) s(-1). Additionally, an upper bound of 1.2 macro nu(e) cm(-2) s(-1) was set for the supernova relic neutrino flux in the energy region E(nu)>19.3 MeV.     

Unexpectedly strong energy stabilization inside the hydrophobic core of small protein rubredoxin mediated by aromatic residues: correlated ab initio quantum chemical calculations

The formation of a hydrophobic core of globular proteins is believed to be the consequence of exterior hydrophobic forces of entropic nature. This, together with the low occurrence of hydrogen bonds in the protein core, leads to the opinion that the energy contribution of core formation to protein folding and stability is negligible. We show that stabilization inside the hydrophobic core of a small protein, rubredoxin, determined by means of high-level correlated ab initio calculations (complete basis set limit of MP2 stabilization energy + CCSD(T) correction term), amounted to approximately 50 kcal/mol. These results clearly demonstrate strong attraction inside a hydrophobic core. This finding may lead to substantial changes in the current view of protein folding. We also point out the inability of the DFT/B3LYP method to describe a strong attraction between studied amino acids.     

On a New Kato Class and Singular Solutions of a Nonlinear Elliptic Equation in Bounded Domains of $$\mathbb{R}^n$$

Using a new form of the 3G-Theorem for the Green function of a bounded domain Ω in , we introduce a new Kato class K(Ω) which contains properly the classical Kato class K_n(Ω). Next, we exploit the properties of this new class, to extend some results about the existence of positive singular solutions of nonlinear differential equations. Mathematics Subject classification (1991): 34B15, 34B27.