Effect of using amino acids in the freeze‑drying of siRNA lipoplexes on gene knockdown in cells after reverse transfection

Calcium (Ca++) and other diffusible ions cannot be preserved for x-ray microanalysis if specimens have been preserved by fixation using formaldehyde, glutaraldehyde or other fixatives. Atoms, such as Ca, are not diffusible when they are bound to something insoluble, such as bone or hard granules in cells which are sometimes mechanisms for detoxifying. Ions, loosely bound elements and other components such as sugars and amino acids are leached out during chemical fixation and dehydration by solvents. This problem was recognized over a hundred years ago by Richard Altmann. An alternative to wet chemical processing is the purely physical method that involves rapidly freezing the specimen and then removing the frozen water by the process of freeze drying, which is the subject of this article.

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Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA

Nature Communications ◽

10.1038/s41467-020-20373-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Howard Gamper ◽

Haixing Li ◽

Isao Masuda ◽

D. Miklos Robkis ◽

Thomas Christian ◽

...

Keyword(s):

Amino Acids ◽

Conformational Change ◽

Molecular Mechanism ◽

Late Stage ◽

Anticodon Loop ◽

Pairing Mechanism ◽

Extra Nucleotide ◽

In Cells

AbstractWhile genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

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Abstract 13879: Ischemic Postconditioning Confers Cardioprotection through Adiponectin-dependent Mitochondrial STAT3 Activation in Mice

Circulation ◽

10.1161/circ.130.suppl_2.13879 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Haobo Li ◽

Michael G Irwin ◽

Zhengyuan Xia

Keyword(s):

Cell Injury ◽

Troponin I ◽

Ischemia Reperfusion Injury ◽

Apoptotic Cell ◽

Ischemia Reperfusion ◽

Cardiac Injury ◽

Systolic Pressure ◽

Gene Knockdown ◽

Area At Risk ◽

In Cells

Introduction: Signal transducer and activator of transcription 3 (STAT3) plays a key role in postconditioning (IPo) mediated protection against myocardial ischemia reperfusion injury, but the mechanism by which IPo activates STAT3 is unknown. Adiponectin (APN), a protein with anti-ischemic properties, activates STAT3. We hypothesized that IPo activates mitochondrial STAT3 (MitoSTAT3) via APN signaling. Methods and Results: Wild type (WT) and APN knockout (KO) mice were either sham operated or subjected to 30 min of coronary artery occlusion followed by 2 hours of reperfusion with or without IPo (3 cycles of 10 seconds reperfusion and 10 seconds reocclusion; n=8/group). At the end of reperfusion, KO mice exhibited more severe myocardial injury evidenced as increased infarct size (% of area at risk) 49.2±2.0 vs WT 39.4±3.5, P <0.01; plasma troponin I (ng/ml): KO 72.8±7.6 vs WT 45.7±4.0, P <0.01; worse cardiac function (lower dP/dt max and end-systolic pressure-volume relation, P <0.05); more severely impaired mitochondrial function (reductions in complex IV and complex V protein expression) and more severe reduction of MitoSTAT3 phosphorylation (activation) at site Ser727, P <0.01. IPo significantly attenuated post-ischemic cardiac injury and dysfunction with a concomitant increase in phosphorylated MitoSTAT3 and attenuation of mitochondrial dysfunction in WT (all P <0.05) but not in KO mice. In cultured cardiac H9C2 cells, hypoxic postconditioning (HPo, 3 cycles of 5 min hypoxia and 5 min reoxygenation) significantly attenuated hypoxia/reoxygenation (HR, 3 hours hypoxia/3 hours reoxygenation) induced cell injury (increased apoptotic cell death as % of HR): HR 100.2±0.4 vs HPo 78.2±4.8, P <0.05) and reduced mitochondrial transmembrane potential (% total cells, HR 37.2±4.9 vs HPo 23.5±3.7, P <0.01). APN, adiponectin receptor 1 (AdipoR1), or STAT3 gene knockdown but not AdipoR2 gene knockdown, respectively, abolished HPo cellular protection (all P <0.05 vs. HPo). APN supplementation (10μg/ml) restored HPo protection in cells with APN knockdown but not in cells with AdipoR1or STAT3 gene knockdown. Conclusion: Adiponectin and AdipoR1 signaling are required for IPo to activate myocardial mitochondrial STAT3 to confer cardioprotection.

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Transport of L-tyrosine by B16/F10 melanoma cells: the effect of the intracellular content of other amino acids

Journal of Cell Science ◽

10.1242/jcs.97.3.479 ◽

1990 ◽

Vol 97 (3) ◽

pp. 479-485

Author(s):

J.R. Jara ◽

J.H. Martinez-Liarte ◽

F. Solano ◽

R. Penafiel

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Initial Rate ◽

Aromatic Amino Acids ◽

Melanoma Cells ◽

Transport Systems ◽

Specific Amino Acid ◽

Intracellular Content ◽

Tyrosine Hydroxylation ◽

In Cells

The uptake of L-Tyr by B16/F10 malignant melanocytes in culture has been studied. These melanoma cells can either be depleted of amino acids by 1 h preincubation in Hanks' isotonic medium or preloaded with a specific amino acid by 1 h preincubation in the same solution containing 2 mM of the amino acid to be preloaded. By means of these pretreatments, it is shown that the rate of L-Tyr uptake is greatly dependent on the content of other amino acids inside the cells. The L-Tyr uptake is higher in cells preloaded with amino acids transported by the L and ASC systems than in cells depleted of amino acids or preloaded with amino acids transported by the A system. It is concluded that L-Tyr is mainly taken up by an exchange mechanism with other amino acids mediated by the L1 system, although the ASC system can also participate in the process. In agreement with that, the homo-exchange performed by cells preloaded with unlabelled L-Tyr is more efficient than any other hetero-exchange, although L-Dopa, the product of tyrosine hydroxylation in melanin synthesis, is almost as efficient as L-Tyr. Apart from aromatic amino acids, melanoma cells preloaded with L-Met and L-His also yield a high initial rate of L-Tyr uptake. The results herein suggest that melanoma cells do not have transport systems specific for L-Tyr, even if this amino acid is needed to carry out the differential pathway of this type of cells, melanosynthesis.

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ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation

Journal of Nutrition ◽

10.1093/jn/nxz309 ◽

2019 ◽

Vol 150 (5) ◽

pp. 1022-1030 ◽

Cited By ~ 5

Author(s):

Dandan Xu ◽

Weiwei Dai ◽

Lydia Kutzler ◽

Holly A Lacko ◽

Leonard S Jefferson ◽

...

Keyword(s):

Amino Acids ◽

Dna Damage ◽

Amino Acid ◽

Protein Kinase ◽

Dna Damage Response ◽

Dependent Manner ◽

Mouse Embryo Fibroblasts ◽

Damage Response ◽

Mtorc1 Activation ◽

In Cells

ABSTRACT Background The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. Objective The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. Methods Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0–16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. Results Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. Conclusions The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

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Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate

Communications Biology ◽

10.1038/s42003-020-01223-6 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 1

Author(s):

Maren Kirstin Schuhmacher ◽

Serap Beldar ◽

Mina S. Khella ◽

Alexander Bröhm ◽

Jan Ludwig ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Histone H3 ◽

Protein Methylation ◽

Sequence Specificity ◽

Sequence Design ◽

Substrate Peptide ◽

Lysine Methyltransferase ◽

In Cells

AbstractSETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.

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Regulation of rRNA synthesis and processing in animal cells. Effect of nucleoside analogues

Biochemical Journal ◽

10.1042/bj2020325 ◽

1982 ◽

Vol 202 (2) ◽

pp. 325-332 ◽

Cited By ~ 7

Author(s):

S Iapalucci-Espinoza ◽

M T Franze-Fernandez

Keyword(s):

Amino Acids ◽

High Rate ◽

Nucleoside Analogues ◽

Rrna Synthesis ◽

Complete Medium ◽

Animal Cells ◽

Rrna Transcription ◽

Ehrlich Ascites ◽

Ehrlich Ascites Cells ◽

In Cells

The nucleoside analogues fluorouridine and fluorodeoxyuridine (both at 100 muM) and 8-azaguanine (at 500 muM) inhibit both rRNA transcription and processing in Ehrlich ascites cells. In BHK21 cells fluorodeoxyuridine has no effect on either rRNA maturation or transcription, whereas toyocamycin (at 2 microM) inhibits both processes in BHK21 cells and Ehrlich ascites cells. The drugs inhibit transcription in cells incubated in the complete medium, but have no effect on the decreased transcription in cells incubated in a medium without amino acids. This lack of effect cannot be explained by an altered uptake of the drugs in the amino acid-starved cells, since maturation of the rRNA precursor is affected in cells incubated in media with or without amino acids. The effect of the drugs on rRNA transcription is not the consequence of the inhibition of protein synthesis. The results lend support to the proposal that rRNA processing and transcription are co-ordinately controlled in cells with a high rate of rRNA synthesis.

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Characterization of Dengue Virus NS4A and NS4B Protein Interaction

Journal of Virology ◽

10.1128/jvi.03453-14 ◽

2015 ◽

Vol 89 (7) ◽

pp. 3455-3470 ◽

Cited By ~ 64

Author(s):

Jing Zou ◽

Xuping Xie ◽

Qing-Yin Wang ◽

Hongping Dong ◽

Michelle Yueqi Lee ◽

...

Keyword(s):

Amino Acids ◽

Dengue Virus ◽

Viral Replication ◽

Transmembrane Domain ◽

Nmr Analysis ◽

Replication Complex ◽

Essential Components ◽

Infected Cells ◽

Er Membrane ◽

In Cells

ABSTRACTFlavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimatedKd(dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach.IMPORTANCEFlavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cells, in cells transiently expressing NS4A and NS4B proteins, orin vitrowith recombinant NS4A and NS4B proteins. We mapped the minimal regions required for the NS4A-NS4B interaction to be amino acids 40 to 76 of NS4A and amino acids 84 to 146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17 to 80 of NS4A and the NS4A amino acids that may participate in the NS4A-NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A-NS4B interaction, demonstrating the biological importance of the NS4A-NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of the NS4A-NS4B interaction could be pursued for flavivirus antiviral development.

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