Synthesis of Chiral N-Nitro-oxazolidin-2-ones and O-(β-Nitraminoalkyl) Carbamates in Liquefied 1,1,1,2-Tetrafluoroethane Medium

AbstractA convenient synthesis of chiral N-nitro-oxazolidin-2-ones by nitration of α-amino acid derived 1,3-oxazolidin-2-ones containing one or two stereogenic centers with dinitrogen pentoxide in liquefied 1,1,1,2-tetrafluoroethane medium has been developed. The obtained N-nitroheterocycles were converted into enantiomerically pure O-(β-nitraminoalkyl) carbamates by treatment with ammonia or amines in the same solvent. The synthesized N-nitro compounds are slightly toxic in vitro to Human Embryonic Kidney 293 (HEK293) cells.

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Inactivation of a Frameshift TSH Receptor Variant Val711Phefs*18 is Due to Acquisition of a Hydrophobic Degron

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgaa772 ◽

2020 ◽

Vol 106 (1) ◽

pp. e265-e272

Author(s):

Chiho Sugisawa ◽

Makoto Ono ◽

Kenichi Kashimada ◽

Tomonobu Hasegawa ◽

Satoshi Narumi

Keyword(s):

Amino Acid ◽

Congenital Hypothyroidism ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Tsh Receptor ◽

Hek293 Cells ◽

Luciferase Reporter ◽

Loss Of Function ◽

Tail Region

Abstract Context Inactivating variants of thyrotropin (thyroid-stimulating hormone; TSH) receptor (TSHR) cause congenital hypothyroidism. More than 60 such variants have been reported so far, most of which were located in the extracellular or transmembrane domain. Objective We report the identification and characterization of a frameshift TSHR variant in the intracytoplasmic C-tail region. Methods Sequencing of TSHR was performed in a patient with congenital hypothyroidism. The functionality of the identified variants was assessed by expressing TSHR in HEK293 cells and measuring TSH-dependent activation of the cAMP–response element-luciferase reporter. A series of systematic mutagenesis experiments were performed to characterize the frameshifted amino acid sequence. Results The proband was heterozygous for a known TSHR variant (p.Arg519His) and a novel frameshift TSHR variant (p.Val711Phefs*18), which removed 54 C-terminal residues and added a 17–amino acid frameshifted sequence. The loss of function of Val711Phefs*18-TSHR was confirmed in vitro, but the function of Val711*-TSHR was found to be normal. Western blotting showed the low protein expression of Val711Phefs*18-TSHR. Fusion of the frameshift sequence to green fluorescent protein or luciferase induced inactivation of them, indicating that the sequence acted as a degron. A systematic mutagenesis study revealed that the density of hydrophobic residues in the frameshift sequence determined the stability. Eight additional frameshift TSHR variants that covered all possible shifted frames in C-tail were created, and another frameshift variant (Thr748Profs*27) with similar effect was found. Conclusions We characterized a naturally occurring frameshift TSHR variant located in C-tail, and provided a unique evidence that hydrophobicity in the C-terminal region of the receptor affects protein stability.

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CRISPR/Cas9 as an antiviral against Orthopoxviruses using an AAV vector

Scientific Reports ◽

10.1038/s41598-020-76449-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Cathryn M. Siegrist ◽

Sean M. Kinahan ◽

Taylor Settecerri ◽

Adrienne C. Greene ◽

Joshua L. Santarpia

Keyword(s):

Targeted Delivery ◽

Model Organism ◽

Host Cells ◽

Hek293 Cells ◽

Viral Titer ◽

Human Embryonic Kidney ◽

Adeno Associated Virus ◽

Guide Rnas ◽

Per Gene

Abstract A vaccine for smallpox is no longer administered to the general public, and there is no proven, safe treatment specific to poxvirus infections, leaving people susceptible to infections by smallpox and other zoonotic Orthopoxviruses such as monkeypox. Using vaccinia virus (VACV) as a model organism for other Orthopoxviruses, CRISPR–Cas9 technology was used to target three essential genes that are conserved across the genus, including A17L, E3L, and I2L. Three individual single guide RNAs (sgRNAs) were designed per gene to facilitate redundancy in rendering the genes inactive, thereby reducing the reproduction of the virus. The efficacy of the CRISPR targets was tested by transfecting human embryonic kidney (HEK293) cells with plasmids encoding both SaCas9 and an individual sgRNA. This resulted in a reduction of VACV titer by up to 93.19% per target. Following the verification of CRISPR targets, safe and targeted delivery of the VACV CRISPR antivirals was tested using adeno-associated virus (AAV) as a packaging vector for both SaCas9 and sgRNA. Similarly, AAV delivery of the CRISPR antivirals resulted in a reduction of viral titer by up to 92.97% for an individual target. Overall, we have identified highly specific CRISPR targets that significantly reduce VACV titer as well as an appropriate vector for delivering these CRISPR antiviral components to host cells in vitro.

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Evaluation of Graphene Oxide Induced Cellular Toxicity and Transcriptome Analysis in Human Embryonic Kidney Cells

Nanomaterials ◽

10.3390/nano9070969 ◽

2019 ◽

Vol 9 (7) ◽

pp. 969 ◽

Cited By ~ 16

Author(s):

Sangiliyandi Gurunathan ◽

Muhammad Arsalan Iqbal ◽

Muhammad Qasim ◽

Chan Hyeok Park ◽

Hyunjin Yoo ◽

...

Keyword(s):

Graphene Oxide ◽

Molecular Mechanisms ◽

Cellular Responses ◽

Hek293 Cells ◽

Single Atom ◽

Ros Generation ◽

Human Embryonic Kidney ◽

Human Embryonic Kidney Cells ◽

In Vitro Investigation

Graphene, a two-dimensional carbon sheet with single-atom thickness, shows immense promise in several nanoscientific and nanotechnological applications, including in sensors, catalysis, and biomedicine. Although several studies have shown the cytotoxicity of graphene oxide in different cell types, there are no comprehensive studies on human embryonic kidney (HEK293) cells that include transcriptomic analysis and an in vitro investigation into the mechanisms of cytotoxicity following exposure to graphene oxide. Therefore, we exposed HEK293 cells to different concentrations of graphene oxide for 24 h and performed several cellular assays. Cell viability and proliferation assays revealed a significant dose-dependent cytotoxic effect on HEK293 cells. Cytotoxicity assays showed increased lactate dehydrogenase (LDH) leakage and reactive oxygen species (ROS) generation, and decreased levels of reduced glutathione (GSH) and increased level of oxidized glutathione indicative of oxidative stress. This detailed mechanistic approach showed that graphene oxide exposure elicits significant decreases in mitochondrial membrane potential and ATP synthesis, as well as in DNA damage and caspase 3 activity. Furthermore, our RNA-Seq analysis revealed that HEK293 cells exposed to graphene oxide significantly altered the expression of genes involved in multiple apoptosis-related biological pathways. Moreover, graphene oxide exposure perturbed the expression of key transcription factors, promoting these apoptosis-related pathways by regulating their downstream genes. Our analysis provides mechanistic insights into how exposure to graphene oxide induces changes in cellular responses and massive cell death in HEK293 cells. To our knowledge, this is the first study describing a combination of cellular responses and transcriptome in HEK293 cells exposed to graphene oxide nanoparticles, providing a foundation for understanding the molecular mechanisms of graphene oxide-induced cytotoxicity and for the development of new therapeutic strategies.

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Electron probe x-ray microanalysis and electron microscopy of amino acid absorption and transport by the small intestine: inhibition by chemical poisons and correlation to the elemental composition of the epithelial cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142311 ◽

1986 ◽

Vol 44 ◽

pp. 134-135

Author(s):

A. J. Tousimis

Keyword(s):

Small Intestine ◽

Amino Acid ◽

Epithelial Cells ◽

Elemental Composition ◽

Isotope Labeling ◽

Structural Components ◽

X Ray ◽

Human Small Intestine ◽

Transport Studies

The elemental composition of amino acids is similar to that of the major structural components of the epithelial cells of the small intestine and other tissues. Therefore, their subcellular localization and concentration measurements are not possible by x-ray microanalysis. Radioactive isotope labeling: I131-tyrosine, Se75-methionine and S35-methionine have been successfully employed in numerous absorption and transport studies. The latter two have been utilized both in vitro and vivo, with similar results in the hamster and human small intestine. Non-radioactive Selenomethionine, since its absorption/transport behavior is assumed to be the same as that of Se75- methionine and S75-methionine could serve as a compound tracer for this amino acid.

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Faculty Opinions recommendation of In vitro biosynthesis of the nonproteinogenic amino acid methoxyvinylglycine.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.733025302.793551891 ◽

2018 ◽

Author(s):

Zixin Deng ◽

Xudong Qu

Keyword(s):

Amino Acid ◽

In Vitro Biosynthesis

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Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19872317 ◽

1987 ◽

Vol 52 (9) ◽

pp. 2317-2325 ◽

Cited By ~ 5

Author(s):

Jan Hlaváček ◽

Jan Pospíšek ◽

Jiřina Slaninová ◽

Walter Y. Chan ◽

Victor J. Hruby

Keyword(s):

Amino Acid ◽

Solid Phase Synthesis ◽

Solid Phase ◽

The Other ◽

Amino Acid Residues ◽

Phase Synthesis ◽

Other Hand ◽

Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

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Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19951229 ◽

1995 ◽

Vol 60 (7) ◽

pp. 1229-1235 ◽

Cited By ~ 2

Author(s):

Ivana Zoulíková ◽

Ivan Svoboda ◽

Jiří Velek ◽

Václav Kašička ◽

Jiřina Slaninová ◽

...

Keyword(s):

Amino Acid ◽

Biological Activities ◽

Residual Activity ◽

Detailed Examination ◽

Amino Acid Residues ◽

Linear Peptide ◽

Zone Electrophoresis ◽

Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

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