scholarly journals Multivalent dendritic polyglycerolamine with arginine and histidine end groups for efficient siRNA transfection

2015 ◽  
Vol 11 ◽  
pp. 763-772 ◽  
Author(s):  
Fatemeh Sheikhi Mehrabadi ◽  
Hanxiang Zeng ◽  
Mark Johnson ◽  
Cathleen Schlesener ◽  
Zhibin Guan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transfection Efficiency ◽  
Endosomal Escape ◽  
Primary Amines ◽  
Ph Responsive ◽  
3T3 Cells ◽  
Sirna Transfection ◽  
Efficient Delivery ◽  
End Groups ◽  
Nih 3T3

The success of siRNA-based therapeutics highly depends on a safe and efficient delivery of siRNA into the cytosol. In this study, we post-modified the primary amines on dendritic polyglycerolamine (dPG-NH2) with different ratios of two relevant amino acids, namely, arginine (Arg) and histidine (His). To investigate the effects from introducing Arg and His to dPG, the resulting polyplexes of amino acid functionalized dPG-NH2s (AAdPGs)/siRNA were evaluated regarding cytotoxicity, transfection efficiency, and cellular uptake. Among AAdPGs, an optimal vector with (1:3) Arg to His ratio, showed efficient siRNA transfection with minimal cytotoxicity (cell viability ≥ 90%) in NIH 3T3 cells line. We also demonstrated that the cytotoxicity of dPG-NH2 decreased as a result of amino acid functionalization. While the incorporation of both cationic (Arg) and pH-responsive residues (His) are important for safe and efficient siRNA transfection, this study indicates that AAdPGs containing higher degrees of His display lower cytotoxicity and more efficient endosomal escape.

scholarly journals rasH mutants deficient in GTP binding.

1986 ◽  
Vol 6 (9) ◽  
pp. 3291-3294 ◽  
Author(s):  
C J Der ◽  
B T Pan ◽  
G M Cooper
Keyword(s):  
Amino Acid ◽  
Binding Affinity ◽  
Binding Proteins ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
3T3 Cells ◽  
Protein Residues ◽  
Gtp Binding ◽  
Nih 3T3 ◽  
P21 Proteins

Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins. Each of the mutant p21 proteins displayed a significant reduction (10- to 5,000-fold) in GTP binding affinity. Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.

scholarly journals The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation

2003 ◽  
Vol 371 (2) ◽  
pp. 395-403 ◽  
Author(s):  
Sharron A.N. BROWN ◽  
Christine M. RICHARDS ◽  
Heather N. HANSCOM ◽  
Sheau-Line Y. FENG ◽  
Jeffrey A. WINKLES
Keyword(s):  
Amino Acid ◽  
Tumour Necrosis Factor ◽  
Tumour Necrosis ◽  
Cytoplasmic Tail ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Necrosis Factor ◽  
Nih 3T3 Cells

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory κBα phosphorylation and transcriptional activation of a nuclear factor-κB (NF-κB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-κB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-κB transcription factor signalling pathway.

Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the nucleus

1988 ◽  
Vol 8 (12) ◽  
pp. 5495-5503
Author(s):  
L Fischer-Fantuzzi ◽  
C Vesco
Keyword(s):  
Amino Acid ◽  
Nuclear Transport ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
3T3 Cells ◽  
Rat Embryo ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We investigated the requisites for, and functional consequences of, the relocation to the nucleus of a transforming nonkaryophilic mutant of the simian virus 40 large T antigen (a natural deletion mutant lacking an internal large-T-antigen domain that includes the signal for nuclear transport). Synthetic oligonucleotides were used to obtain gene variants with one or more copies of the signal-specifying sequence inserted near the gene 3' end, in a region dispensable for the main large-T-antigen functions. The analysis of stable transfectant populations showed that mouse NIH 3T3 cells, rat embryo fibroblasts, and simian CS cells (a subclone of CV1 cells) differed considerably in their ability to localize some variant molecules into the nucleus. CS cells were always the most efficient, and NIH 3T3 cells were the least efficient. The nuclear localization improved either with reiteration of the signal or with a left-flank modification of the signal amino acid context. Three signals appeared to be necessary and sufficient, even in NIH 3T3 cells, to obtain a nuclear accumulation comparable to that of wild-type simian virus 40 large T antigen; other signal-cell combinations caused a large variability in subcellular localization among cells of the same population, as if the nuclear uptake of some molecules depended on individual cell states. The effect of the modified location on the competence of the protein to alter cell growth was examined by comparing the activity of variants containing either the normal signal or a signal with a mutation (corresponding to large-T-antigen amino acid 128) that prevented nuclear transport. It was found that the nuclear variant was slightly more active than the cytoplasmic variants in rat embryo fibroblasts and NIH 3T3 cells and was notably less active in CS cells.

rasH mutants deficient in GTP binding

1986 ◽  
Vol 6 (9) ◽  
pp. 3291-3294
Author(s):  
C J Der ◽  
B T Pan ◽  
G M Cooper
Keyword(s):  
Amino Acid ◽  
Binding Affinity ◽  
Binding Proteins ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
3T3 Cells ◽  
Protein Residues ◽  
Gtp Binding ◽  
Nih 3T3 ◽  
P21 Proteins

Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins. Each of the mutant p21 proteins displayed a significant reduction (10- to 5,000-fold) in GTP binding affinity. Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. II. Chloride and amino acid fluxes

1997 ◽  
Vol 272 (6) ◽  
pp. C1804-C1809 ◽  
Author(s):  
J. Moran ◽  
D. Miranda ◽  
C. Pena-Segura ◽  
H. Pasantes-Morales
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Regulatory Volume Decrease ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
3T3 Cells ◽  
P Glycoprotein ◽  
Arachidonic Acids ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The osmolyte function of amino acids and Cl in native NIH/3T3 cells not expressing the P-glycoprotein was examined by investigating the free amino acid concentration and the swelling-activated efflux of [3H]taurine, as representative of amino acids, and of 125I, as a tracer for Cl. Taurine and 125I efflux was activated by 20 and 30% hyposmotic solutions. At 50% hyposmotic solutions, the osmolyte pool was essentially depleted. The Cl channel blockers 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid inhibited the release of the two osmolytes by 80-95%. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (400 microM) decreased the efflux of taurine 80% without affecting that of 125I. Linolenic and arachidonic acids (5-20 microM) showed a concentration-dependent inhibitory effect on taurine and 125I fluxes. Omission of Ca decreased osmolyte fluxes by 16%. Verapamil inhibited the osmolyte release only at 500 microM. Nimodipine at 25 and 50 microM decreased the release of [3H]taurine and 125I by approximately 60 and 80%, respectively, but this effect was independent of the presence of extracellular Ca. These results indicate that amino acids and Cl function as osmolytes during regulatory volume decrease in native NIH/ 3T3 cells.

scholarly journals Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the nucleus.

1988 ◽  
Vol 8 (12) ◽  
pp. 5495-5503 ◽  
Author(s):  
L Fischer-Fantuzzi ◽  
C Vesco
Keyword(s):  
Amino Acid ◽  
Nuclear Transport ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
3T3 Cells ◽  
Rat Embryo ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We investigated the requisites for, and functional consequences of, the relocation to the nucleus of a transforming nonkaryophilic mutant of the simian virus 40 large T antigen (a natural deletion mutant lacking an internal large-T-antigen domain that includes the signal for nuclear transport). Synthetic oligonucleotides were used to obtain gene variants with one or more copies of the signal-specifying sequence inserted near the gene 3' end, in a region dispensable for the main large-T-antigen functions. The analysis of stable transfectant populations showed that mouse NIH 3T3 cells, rat embryo fibroblasts, and simian CS cells (a subclone of CV1 cells) differed considerably in their ability to localize some variant molecules into the nucleus. CS cells were always the most efficient, and NIH 3T3 cells were the least efficient. The nuclear localization improved either with reiteration of the signal or with a left-flank modification of the signal amino acid context. Three signals appeared to be necessary and sufficient, even in NIH 3T3 cells, to obtain a nuclear accumulation comparable to that of wild-type simian virus 40 large T antigen; other signal-cell combinations caused a large variability in subcellular localization among cells of the same population, as if the nuclear uptake of some molecules depended on individual cell states. The effect of the modified location on the competence of the protein to alter cell growth was examined by comparing the activity of variants containing either the normal signal or a signal with a mutation (corresponding to large-T-antigen amino acid 128) that prevented nuclear transport. It was found that the nuclear variant was slightly more active than the cytoplasmic variants in rat embryo fibroblasts and NIH 3T3 cells and was notably less active in CS cells.

scholarly journals THE COMPOSITION AND AMINO ACID END-GROUPS OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASES

1953 ◽  
Vol 203 (2) ◽  
pp. 575-582 ◽  
Author(s):  
Sidney F. Velick ◽  
Sidney Udenfriend
Keyword(s):  
Amino Acid ◽  
End Groups
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