Latex Fractionation by Sedimentation and Colloidal Crystallization: The Case of Poly(styrene-co-acrylamide)

Poly(styrene-co-acrylamide) (PS-AAM) latex was prepared, fractionated by sedimentation under gravity, and characterized by PCS, infrared spectra, secondary and backscattered electron imaging in the scanning electron microscope, and electron spectroscopy imaging in an analytical transmission electron microscope. Three latex fractions were obtained. The lower fraction was opalescent and its particles were the more uniform, concerning size, chemical composition, and topochemical features. This lower fraction was still further fractionated by zonal centrifugation in a density gradient, yielding two fractions with similar macrocrystal-forming abilities but different sizes and chemical compositions. These results confirm those previously obtained for the PS-HEMA latex. Copyright 2000 Academic Press.     

Antisense-mediated isoform switching of steroid receptor coactivator-1 in the central nucleus of the amygdala of the mouse brain

Background

Antisense oligonucleotide (AON)-mediated exon skipping is a powerful tool to manipulate gene expression. In the present study we investigated the potential of exon skipping by local injection in the central nucleus of the amygdala (CeA) of the mouse brain. As proof of principle we targeted the splicing of steroid receptor coactivator-1 (SRC-1), a protein involved in nuclear receptor function. This nuclear receptor coregulator exists in two splice variants (SRC-1a and SRC-1e) which display differential distribution and opposing activities in the brain, and whose mRNAs differ in a single SRC-1e specific exon.

Methods

For proof of principle of feasibility, we used immunofluorescent stainings to study uptake by different cell types, translocation to the nucleus and potential immunostimulatory effects at different time points after a local injection in the CeA of the mouse brain of a control AON targeting human dystrophin with no targets in the murine brain. To evaluate efficacy we designed an AON targeting the SRC-1e-specific exon and with qPCR analysis we measured the expression ratio of the two splice variants.

Results

We found that AONs were taken up by corticotropin releasing hormone expressing neurons and other cells in the CeA, and translocated into the cell nucleus. Immune responses after AON injection were comparable to those after sterile saline injection. A successful shift of the naturally occurring SRC-1a:SRC-1e expression ratio in favor of SRC-1a was observed, without changes in total SRC-1 expression.

Conclusions

We provide a proof of concept for local neuropharmacological use of exon skipping by manipulating the expression ratio of the two splice variants of SRC-1, which may be used to study nuclear receptor function in specific brain circuits. We established that exon skipping after local injection in the brain is a versatile and useful tool for the manipulation of splice variants for numerous genes that are relevant for brain function.     

Lowering the concentration limits of detection by on-line solid-phase extraction-capillary electrophoresis-electrospray mass spectrometry

The use of solid-phase extraction coupled on-line to capillary electrophoresis using electrospray mass spectrometry detection (SPE-CE-ESI-MS) is described for the analysis of peptides in dilute solutions. A SPE microcartridge or analyte concentrator containing C(18) derivatized silica particles as the extraction sorbent was easily constructed near the inlet of the separation capillary using commercially available materials. The reversed-phase sorbent selectively retained the target peptides, enabling large volumes of the sample to be introduced (>100muL). The captured analytes were eluted in a small volume of an appropriate solution (20-50nL). This resulted in sample clean-up and concentration enhancement, with minimum sample handling. As the SPE-CE conditions were compatible with on-line ESI-MS detection, the potential for identifying and characterizing the preconcentrated analytes by SPE-CE-ESI-MS using a sheath-flow CE-ESI-MS interface is also shown. Using separation electrolytes containing N-[carbamoylmethyl]-2-aminoethanesulfonic acid (ACES) at pH 7.4, an elution plug of 80:20 (v/v) (25mM of formic acid in MeCN):H(2)O and a sheath liquid of 20mM of acetic acid in 50:50 (v/v) methanol:H(2)O the concentration limits of detection for the analyzed peptides in the positive ion mode were lowered to nanogram per milliliter levels. The systematic optimization of the operational parameters involved in the development of the SPE-CE method is described in detail, in order to promote robust and quantitative SPE-CE-ESI-MS analysis and facilitate the widespread use of the technique.     

Transfer measurements for the Ti+Ni systems at near barrier energies

Large enhancements have been observed in the sub-barrier fusion cross sections for Ti+Ni systems in our previous studies. Coupled channel calculations incorporating couplings to 2⁺ and 3⁻ states failed to explain these enhancements completely. A possibilty of transfer channels contributing to the residual enhancements had been suggested. In order to investigate the role of relevant transfer channels, measurements of one- and two-nucleon transfer were carried out for ^46,48Ti+⁶¹Ni systems. The present paper gives the results of these studies.