Cholesterol conjugated oligonucleotide and LNA: A comparison of cellular and nuclear uptake by Hep2 cells enhanced by Streptolysin-O

Antisense and antigene oligonucleotides (ONs) are attractive drugs for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Adding various lipophilic conjugates to the ON improves intracellular delivery as has been previously reported.We studied the cellular delivery of various ON modifications, as well as their cytosolic and nuclear distribution in mammalian Hep2-EGFP-NLS cell line. We compared uptake efficacy of ON and LNA, both conjugated with cholesterol at the 5′ end. All ONs were 3′ labeled with fluorescent Cy5 dye. We made a comparison of the ONs uptake efficacy and the kinetics, both adding ONs to the culture medium, and using streptolysin-O (SL-O) permeabilization.The cellular uptake of each ON used in this study was visualized by fluorescent microscopy. We confirmed the results by FACS analysis. We determined the ratio between initial ON-chol concentration (0.4 μM) and the final amount in nucleus.SL-O can highly improve kinetics of ON delivery; not only into the cytoplasm but also to the nucleus, the presumed site of antigene ON action. The most effective nuclear uptake was observed when ON conjugated with cholesterol (ON-chol) and SL-O was used. Nuclear distribution of ON was reached within few minutes. In contrast, ON simply added to the medium reached cytoplasm only and the process of delivery took several hours. (Mol Cell Biochem 276: 61–69, 2005)     

Stimulation of the DNA unwinding activity of human DNA helicase II/Ku by phosphorylation

The Ku autoantigen is a heterodimeric protein of 70- and 83-kDa subunits, endowed with duplex DNA end-binding capacity and DNA helicase activity (Human DNA Helicase II, HDH II). HDH II/Ku is well established as the DNA binding component, the regulatory subunit as well as a substrate for the DNA-dependent protein kinase DNA-PK, a complex involved in the repair of DNA double-strand breaks and in V(D)J recombination in eukaryotes. The effects of phosphorylation by this kinase on the helicase activity of Escherichia coli-produced HDH II/Ku were studied. The rate of DNA unwinding by recombinant HDH II/Ku heterodimer is stimulated at least fivefold upon phosphorylation by DNA-PK_cs. This stimulation is due to the effective transfer of phosphate residues to the helicase rather than the mere presence of the complex. In vitro dephosphorylation of HeLa cellular HDH II/Ku caused a significant decrease in the DNA helicase activity of this enzyme.     

Angiotensin II immunoreactivity in push-pull perfusates from the paraventricular nucleus

Push-pull perfusion of the hypothalamic paraventricular nucleus in sodium pentobarbital anesthetized Sprague-Dawley rats indicates the release of angiotensin II-immunoreactive material in this area. Attempts to demonstrate a neuronal origin of this material by chemical depolarization with perfusate containing either 40 or 120 mM K+ were unsuccessful. However, this material does appear to be of central origin since intravenous infusion of arginine-vasopressin, a similar sized peptide, did not result in the appearance of increased levels of this substrate in the perfusate, indicating that the integrity of the blood-brain barrier was not compromised by the perfusion.     

Controlling pH in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics

Background  

There are significant differences in the culture conditions between small-scale screenings and large-scale fermentation processes. Production processes are usually conducted in fed-batch cultivation mode with active pH-monitoring and control. In contrast, screening experiments in shake flasks are usually conducted in batch mode without active pH-control, but with high buffer concentrations to prevent excessive pH-drifts. These differences make it difficult to compare results from screening experiments and laboratory and technical scale cultivations and, thus, complicate rational process development. In particular, the pH-value plays an important role in fermentation processes due to the narrow physiological or optimal pH-range of microorganisms. To reduce the differences between the scales and to establish a pH-control in shake flasks, a newly developed easy to use polymer-based controlled-release system is presented in this paper. This system consists of bio-compatible silicone discs embedding the alkaline reagent Na₂CO₃. Since the sodium carbonate is gradually released from the discs in pre-determined kinetics, it will ultimately compensate the decrease in pH caused by the biological activity of microorganisms.     

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.     

Identification and expression of Ima,a novel Ral-interacting Drosophila protein

We report the identification of Ima, a novel Drosophila MAGUK-like protein, which contains two WW and four PDZ protein interaction domains and interacts with the small GTPase dRal in the yeast two-hybrid system and pull-down assays. The gene is expressed in distinct spatiotemporal patterns throughout embryonic development. Overexpression of Ima interferes with normal Drosophila development, indicating that the gene functions in a tissue specific manner.     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

Book Reviews

The findings in and experiences with 19 consecutive patients subjected to open mitral valve surgery are described. All patients underwent a right-heart catheterization. In order to exclude multivalvular heart disease, a left-heart catheterization was performed in 10 patients and angiographic studies of the aortic valve area in 12. Pulmonary function studies were performed on 11 patients. The FEV (0.75 sec.) and the MMFR were found to correlate well with existing pulmonary reserves.Fourteen of the 19 patients subjected to open mitral valve surgery survived the operation and have been followed up for three months to 2½ years. Four patients had a mitral valvuloplasty. Three of these four have deteriorated and will require a valvular replacement. Ten of 15 patients subjected to a mitral valve replacement are alive; five of these 10 have had signs and symptoms indicating peripheral embolization. Mitral valvuloplasty is preferable to a valvular replacement as far as embolic complications are concerned, while valvular replacements result in a more perfect and lasting hemodynamic repair. The high incidence of peripheral embolization following valvular replacements focuses attention on the need for improved mitral valve prostheses.     

Bacterial Metabolism of 2,6-Xylenol

Strain DM1, a Mycobacterium sp. that utilizes 2,6-xylenol, 2,3,6-trimethylphenol, and o-cresol as sources of carbon and energy, was isolated. Intact cells of Mycobacterium strain DM1 grown with 2,6-xylenol cooxidized 2,4,6-trimethylphenol to 2,4,6-trimethylresorcinol. 4-Chloro-3,5-dimethylphenol prevents 2,6-xylenol from being totally degraded; it was quantitatively converted to 2,6-dimethylhydroquinone by resting cells. 2,6-Dimethylhydroquinone, citraconate, and an unidentified metabolite were detected as products of 2,6-xylenol oxidation in cells that were partially inactivated by EDTA. Under oxygen limitation, 2,6-dimethylhy-droquinone, citraconate, and an unidentified metabolite were released during 2,6-xylenol turnover by resting cells. Cell extracts of 2,6-xylenol-grown cells contained a 2,6-dimethylhydroquinone-converting enzyme. When supplemented with NADH, cell extracts catalyzed the reduction of 2,6-dimethyl-3-hydroxyquinone to 2,6-dimethyl-3-hydroxyhydroquinone. Since a citraconase was also demonstrated in cell extracts, a new metabolic pathway with 2,6-dimethyl-3-hydroxyhydroquinone as the ring fission substrate is proposed.