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21.
Joshua Seaberg Nicholas Flynn Amanda Cai Joshua D. Ramsey 《Biotechnology and bioengineering》2020,117(8):2504-2515
Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l -lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins. 相似文献
22.
23.
Dimitrij Lang H.Thomas Steely Jr. Chia-Yi Kao Nicholas T. Ktistakis 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1987,910(3)
The contour lengths of linear, double-stranded (ds) RNAs from mycovirus PcV and Pseudomonas bacteriophage ø6 have been measured with samples prepared for the electron microscope from 0.05 to 0.5 M NH4Cl solutions. A linear dependence of contour length on the logarithm of ionic strength was found and compared with that of dsDNA (pBR322, linearized and open-circular forms). Conditions for molecular weight determinations of any natural dsRNA by electron microscopy have been established, and the method has been calibrated with ø6 dsRNA of known nucleotide sequence. The results imply that dsRNA in 0.20 M NH4Cl solution has a rise per basepair of 0.271 nm, which is shorter than that in the A-conformation (4%) and in the A′-conformation (10%). The thermal behavior of dsRNA in terms of melting temperature and exhibition of fine structure of melting curves was found to be generally similar to that of dsDNA, as expected from the literature. Folding of dsRNA in ethanolic solution was similar to that of dsDNA. However, in contrast to dsDNA, coiled coils could not be induced by ethanol, which is consistent with dsRNA being stiffer than dsDNA. Concerning dsDNA, the results show that a contraction in rise per basepair by 0.1 nm is coupled with an increase in the winding angle between basepairs by 0.47°, as qualitatively predicted by polyelectrolyte theory. 相似文献
24.
Carmen A. Mannella Joachim Frank Nicholas Delihas 《Journal of molecular evolution》1987,24(3):228-235
Summary Correspondence analysis (a form of multivariate statistics) applied to 74 5S ribosomal RNA sequences indicates that the sequences are interrelated in a systematic, nonrandom fashion. Aligned sequences are represented as vectors in a 5N-dimensional space, where N is the number of base positions in the 5S RNA molecule. Mutually orthogonal directions (called factor axes) along which intersequence variance is greatest are defined in this hyperspace. Projection of the sequences onto planes defined by these factorial directions reveals clustering of species that is suggestive of phylogenetic relationships. For each factorial direction, correspondence analysis points to regions of importance, i.e., those base positions at which the systematic changes occur that define that particular direction. In effect, the technique provides a rapid determination of group-specific signatures. In several instances, similarities between sequences are indicated that have only recently been inferred from visual base-to-base comparisons. These results suggest that correspondence analysis may provide a valuable starting point from which to uncover the patterns of change underlying the evolution of a macromolecule, such as 5S RNA. 相似文献
25.
Differences between transfer RNA molecules 总被引:7,自引:0,他引:7
Computer-assisted comparisons of 67 tRNA sequences that function in Escherichia coli or Salmonella typhimurium were used to identify single and multiple nucleotide positions that maximally distinguish the 20 amino acid acceptor groups. Positions in the anticodon were identified most frequently, as expected from the decoding function of this region of the tRNA. The biological function, if any, of positions outside the anticodon may include specificity for aminoacyl-tRNA synthetase enzymes. 相似文献
26.
Synthetic peptide SH624 (SHHPARTAHYGSLPQK), residues 59–74 of human myelin basic protein (MBP) was found to be encephalitogenic in the rabbit. Four antisera raised, against the peptide were employed in a liquid-phase equilibrium competitive radioimmunoassay with a series of synthetic peptide analogs of the region to probe the structural requirements of the B-cell determinant subsumed within SH624. The cross-reactivities of the four antisera with intact MBP were also examined. Immunochemical analyses of the four antisera suggested specificities directed against a conformational determinant dependent upon residues from the more phylogenetically conserved carboxyl C-terminal region, residues 65–74 (TAHYGSLPQK) of the synthetic immunogen. Peptide analogs shorter than SH624 from the C-terminal end showed no cross-reactivity with any of the reagent antisera while analogs shorter from the N-terminal end and including the encephalitogenic sequence TTHYGSLPQK, as well as, HYGSLPQK were reactive under equilibrium competitive conditions. SH624-reactive antibodies, cross-reactive with purified heterologous MBPs from 10 different species were also identified in all four reagent antisera. The results of these experiments support previous investigations demonstrating the accessibility of the encephalitogenic 65–74 region in intact MBP. They also underscore the importance of B-cell recognition of organ specific antigenic determinants with respect to MBP immunology and, in particular, the recognition of autoreactive determinants in the neighborhood of encephalitogenic centers. 相似文献
27.
28.
Evidence is presented that although many proteins from the fronds of Lemna minor L. undergo enhanced degradation during osmotic stress, ribulose-1,5-bisphosphate carboxylase (RuBPCase) is not degraded. Instead RuBPCase is converted in a series of steps to a very high-molecular-weight form. The first step involves the induction of an oxidase system which after 24 h of stress converts RuBPCase to an acidic and catalytically inactive form. Subsequently, the oxidised RuBPCase protein is gradually polymerized to a number of very large aggregates (molecular weight of several million).The conversion of RuBPCase to a high-molecular-weight form appears to be correlated with (i) a reduction in the number of-SH residues and (ii) the susceptibility to in-vitro proteolysis. Indeed, the number of-SH groups per RuBPCase molecule decreases from 89 in the native enzyme to 54 and 22 in the oxidised and polymerized forms, respectively. On the other hand, the oxidised enzyme is more susceptible to in-vitro proteolysis than the native form. However, it is the polymerized form of RuBPCase which is particularly susceptible to in-vitro proteolysis.Western-blotting experiments and anti-ubiquitin antibodies were used to detect the presence of ubiquitin conjugates in extracts from osmotically stressed Lemna fronds. The possible involvement of ubiquitin in the formation of the aggregates is discussed.Abbreviations DTT
dithiothreitol
- EDTA
ethylenediamine-tetraacetic acid
- FPLC
fast protein liquid chromatography
- kDa
kilodaltons
- PAGE
polyacrylamide gel electrophoresis
- PMSF
phenylmethylsulphonyl fluoride
- RuBPCase
ribulose bisphosphate carboxylase
- SDS
sodium dodecyl sulphate
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
29.
Cyclic nucleotides play a central role in the modulation of ion channels in a variety of tissues, including the heart. In order to determine the possible role of cyclic GMP (cGMP) in the regulation of the background K channel activity of cardiac cells, the effect of 8-Br-cGMP on the inwardly-rectifying K channels of cultured ventricular myocytes from embryonic chick hearts was examined. 8-Br-cGMP (10-4 to 10-3 M) inhibited these single channel currents within 3 to 10 min. Spontaneous recovery of the currents occurred with prolonged ( 15 min) exposure to 8-Br-cGMP, but this recovery was accompanied by altered channel behavior. Thus, a new long-lasting open state of the channel appeared, in addition to the open state observed prior to 8-Br-cGMP addition. Superfusion of the cells with the muscarinic agonist carbamylcholine (10-5 M) also resulted in inhibition of the currents, which suggests that the cGMP-mediated inhibition of these channels may occur under physiological conditions. Thus, it appears that cGMP may be an important modulator of the background K conductance (and excitability) of cardiac cells. 相似文献
30.
Xiang-Qin Liu Jonathan P. Hosler John E. Boynton Nicholas W. Gillham 《Plant molecular biology》1989,13(4):385-394
Previous studies have identified a set of highly phosphorylated proteins of 23–25 kDa accumulated during normal embryogenesis of Zea mays L. and which disappear in early germination. They can be induced precociously in embryos by abscisic acid (ABA) treatment. Here the synthesis and accumulation of this group of proteins and their corresponding mRNAs were examined in ABA-deficient viviparous embryos at different developmental stages whether treated or not with ABA, and in water-stressed leaves of both wild-type and viviparous mutants.During embryogenesis and precocious germination of viviparous embryos the pattern of expression of the 23–25 kDa proteins and mRNAs closely resembles that found in non-mutant embryo development. They are also induced in young viviparous embryos by ABA treatment. In contrast, leaves of ABA-deficient mutants fail to accumulate mRNA in water stress, yet do respond to applied ABA. In water-stressed leaves of wild type plants the mRNAs are induced and translated into 4 proteins with a molecular weight and isoelectric point identical to those found in embryos.These results indicate that the 23–25 kDa protein set is a new member of the recently described class or proteins involved in generalized plant ABA responses.The different pattern of expression for the ABA-regulated 23–25 kDa proteins and mRNAs found in embryo and in vegetative tissues of viviparous mutants is discussed. 相似文献