Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.
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PMID:Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes. 1698 30

Senescence is a permanent growth arrest that restricts the lifespan of primary cells in culture, and represents an in vitro model for aging. Senescence functions as a tumor suppressor mechanism that can be induced independent of replicative crisis by diverse stress stimuli. RNase-L mediates antiproliferative activities and functions as a tumor suppressor in prostate cancer, therefore, we examined a role for RNase-L in cellular senescence and aging. Ectopic expression of RNase-L induced a senescent morphology, a decrease in DNA synthesis, an increase in senescence-associated beta-galactosidase activity, and accelerated replicative senescence. In contrast, senescence was retarded in RNase-L-null fibroblasts compared with wild-type fibroblasts. Activation of endogenous RNase-L by 2-5A transfection induced distinct senescent and apoptotic responses in parental and Simian virus 40-transformed WI38 fibroblasts, respectively, demonstrating cell type specific differences in the antiproliferative response to RNase-L activation. Replicative senescence is a model for in vivo aging; therefore, genetic disruption of senescence effectors may impact lifespan. RNase-L-/- mice survived 31.7% (P<0.0001) longer than strain-matched RNase-L+/+ mice providing evidence for a physiological role for RNase-L in aging. These findings identify a novel role for RNase-L in senescence that may contribute to its tumor suppressive function and to the enhanced longevity of RNase-L-/- mice.
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PMID:Role of 2-5A-dependent RNase-L in senescence and longevity. 1713 Aug 39

Type II restriction-modification (R-M) systems are composed of linked restriction endonuclease and modification methyltransferase genes and serve as barriers to horizontal gene transfer even though they are mobile in themselves. Their products kill host bacterial cells that have lost the R-M genes, a process that helps to maintain the frequency of the R-M systems in the viable cell population. Their establishment and maintenance in a bacterial host are expected to involve fine regulation of their gene expression. In the present study, we analyzed transcription of the modification gene and its regulation within the EcoRI R-M system. Northern blotting revealed that the downstream ecoRIM gene is transcribed as a monocistronic mRNA and as part of a larger bicistronic mRNA together with the upstream ecoRIR gene. Primer extension, RNase protection, and mutational analysis using lacZ gene fusions identified two overlapping promoters for ecoRIM gene transcription within the ecoRIR gene. Further mutational analysis revealed that two upstream AT-rich elements within the ecoRIR gene, "AATAAA" and "ATTATAAATATA," function as negative regulators of these promoters. Simultaneous substitution of these two elements resulted in a four-fold increase in beta-galactosidase activity and a five-fold increase in transcript levels as measured by RNase protection assay. RNA measurements of the ecoRIM transcript suggested that these elements decreased ecoRIM expression by interfering with transcription initiation of the ecoRIM promoters. Possible roles for these ecoRIM promoters and their negative regulators in the EcoRI R-M system are discussed.
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PMID:Regulation of the EcoRI restriction-modification system: Identification of ecoRIM gene promoters and their upstream negative regulators in the ecoRIR gene. 1761 69


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