scholarly journals Post-translational incorporation of the antiproliferative agent azatyrosine into the C-terminus of α-tubulin

2003 ◽  
Vol 375 (1) ◽  
pp. 121-129 ◽  
Author(s):  
Silvia A. PURRO ◽  
C. Gastón BISIG ◽  
María A. CONTIN ◽  
Héctor S. BARRA ◽  
Carlos A. ARCE
Keyword(s):  
Cultured Cells ◽  
In Vivo Studies ◽  
Brain Extract ◽  
Similar Degree ◽  
C6 Cells ◽  
Post Translational Modification ◽  
Α Subunit ◽  
C Terminus

Detyrosination/tyrosination of tubulin is a post-translational modification that occurs at the C-terminus of the α-subunit, giving rise to microtubules rich in either tyrosinated or detyrosinated tubulin which coexist in the cell. We hereby report that the tyrosine analogue, azatyrosine, can be incorporated into the C-terminus of α-tubulin instead of tyrosine. Azatyrosine is structurally identical to tyrosine except that a nitrogen atom replaces carbon-2 of the phenolic group. Azatyrosine competitively excluded incorporation of [14C]tyrosine into tubulin of soluble brain extract. A newly developed rabbit antibody specific to C-terminal azatyrosine was used to study incorporation of azatyrosine in cultured cells. When added to the culture medium (Ham's F12K), azatyrosine was incorporated into tubulin of glioma-derived C6 cells. This incorporation was reversible, i.e. after withdrawal of azatyrosine, tubulin lost azatyrosine and reincorporated tyrosine. Azatyrosinated tubulin self-assembled into microtubules to a similar degree as total tubulin both in vitro and in vivo. Studies by other groups have shown that treatment of certain types of cultured cancer cells with azatyrosine leads to reversion of phenotype to normal, and that administration of azatyrosine into animals harbouring human proto-oncogenic c-Ha-ras prevents tumour formation. These interesting observations led us to study this phenomenon in relation to tubulin status. Under conditions in which tubulin was mostly azatyrosinated, C6 cells remained viable but did not proliferate. After 7–10 days under these conditions, morphology changed from a fused, elongated shape to a rounded soma with thin processes. Incorporation of azatyrosine into the C-terminus of α-tubulin is proposed as one possible cause of reversion of the malignant phenotype.

scholarly journals New Soluble Angiopoietin Analog of C4BP-ANG1 Prevents Pathological Vascular Leakage

2020 ◽  
Author(s):  
Pan Liu ◽  
Michael Ryczko ◽  
Xinfang Xie ◽  
Aftab Taiyab ◽  
Heather Sheardown ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Vascular Inflammation ◽  
Vascular Leakage ◽  
Organ Injury ◽  
Complement Protein ◽  
C Terminus ◽  
Vascular Stability ◽  
Important Regulator

AbstractVascular leak is a key driver of organ injury in diseases such as Acute Respiratory Distress Syndrome caused by viruses, including COVID-19. Strategies that reduce enhanced permeability and vascular inflammation are promising therapeutic targets. Activation of the Angiopoietin-1 (Angpt1)-Tie2 tyrosine kinase signaling pathway is an important regulator of vascular quiescence. Here we describe the design and construction of a new soluble ANGPT1 mimetic that is a potent activator of endothelial Tie2 in vitro and in vivo. Using a chimeric fusion strategy, we replaced the extracellular matrix (ECM) binding and oligomerization domain of ANGPT1 with a heptameric scaffold derived from the C-terminus of serum complement protein C4-binding protein α (C4BP). We refer to this new fusion protein biologic as C4BP-ANG1, which forms a stable heptamer and induces TIE2 phosphorylation in cultured cells, and in the lung following i.v. injection of mice. Injection of C4BP-ANG1 ameliorates VEGF- and lipopolysaccharide-induced vascular leakage, in keeping with the known functions of Angpt1-Tie2 in maintaining quiescent vascular stability, and therefore is a promising candidate treatment for inflammatory endothelial dysfunction.

The VSL#3 probiotic formula induces mucin gene expression and secretion in colonic epithelial cells

2007 ◽  
Vol 292 (1) ◽  
pp. G315-G322 ◽  
Author(s):  
C. Caballero-Franco ◽  
K. Keller ◽  
C. De Simone ◽  
K. Chadee
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
In Vivo Studies ◽  
Lactobacillus Species ◽  
Mucin Secretion ◽  
Colonic Epithelial Cells ◽  
Dnase Treatment

Several studies have stressed the importance of the microbiota in the maintenance of the gastrointestinal epithelium. Administration of probiotic bacteria, supplements composed of microbiota constituents, was previously shown to diminish symptoms in patients suffering from inflammatory bowel diseases. This raises the possibility that probiotics may play an active role in enhancing the intestinal barrier at the mucosal surface. In this study, we investigated whether the clinically tested VSL#3 probiotic formula and/or its secreted components can augment the protective mucus layer in vivo and in vitro. For in vivo studies, Wistar rats were orally administered the probiotic mixture VSL#3 on a daily basis for seven days. After treatment, basal luminal mucin content increased by 60%. In addition, we exposed isolated rat colonic loops to the VSL#3 probiotic formula, which significantly stimulated colonic mucin (MUC) secretion and MUC2 gene expression; however, MUC1 and MUC3 gene expression were only slightly elevated. The effect of the VSL#3 mucin secretagogue was also tested in vitro by use of LS 174T colonic epithelial cells. In contrast to the animal studies, cultured cells incubated with VSL#3 bacteria did not exhibit increased mucin secretion. However, the bacterial secreted products contained in the conditioned media stimulated a remarkable mucin secretion effect. Among the three bacterial groups ( Lactobacilli, Bifidobacteria, and Streptococci) contained in VSL#3, the Lactobacillus species were the strongest potentiator of mucin secretion in vitro. A preliminary characterization of the putative mucin secretagogue suggested that it was a heat-resistant soluble compound, which is not sensitive to protease and DNase treatment. These findings contribute to a better understanding of the complex and beneficial interaction between colonic epithelial cells and intestinal bacteria.

Hyperoxic stress elevates p52(PAI-1) mRNA abundance in cultured cells and adult rat pulmonary tissue

1993 ◽  
Vol 265 (2) ◽  
pp. L121-L126
Author(s):  
J. E. White ◽  
M. P. Ryan ◽  
M. F. Tsan ◽  
P. J. Higgins
Keyword(s):  
Cultured Cells ◽  
Rat Kidney ◽  
Mrna Abundance ◽  
In Vivo Studies ◽  
Mrna Levels ◽  
Adult Rats ◽  
Pulmonary Tissue ◽  
Pai 1

Hyperoxic stress alters expression of genes involved in extracellular matrix (ECM) remodeling. To identify novel ECM-associated gene products positively regulated by hyperoxia, rat kidney cells were exposed to 95% O2, and the complement of [35S]methionine-labeled, saponin-resistant, ECM-associated proteins was compared with normoxic controls. O2-stressed cells accumulated significantly greater ECM levels (approximately 3- to 4-fold that of control cells) of a 52-kDa glycoprotein (p52), recently identified as the matrix form of plasminogen activator inhibitor type 1 (PAI-1) (P.J. Higgins, P. Chaudhari, and M.P. Ryan. Biochem. J. 273: 651-658, 1991; P. J. Higgins, M. P. Ryan, R. Zeheb, T. D. Gelehrter, P. Chaudhari. J. Cell. Physiol. 143:321-329, 1990), which peaked at 48 h of exposure. Hyperoxia-associated increases in ECM p52(PAI-1) content reflected parallel elevations in p52(PAI-1) mRNA abundance. Similar results were obtained using secondary cultures of rat pulmonary fibroblasts. This 48-h period of maximal hyperoxia-induced p52(PAI-1) expression in vitro was used to design subsequent in vivo studies. Adult rats were exposed to 99% O2 for 24–50 h, and RNA was extracted from the pulmonary tissue of stressed and control animals. A 5- to 8-fold and 6- to 15-fold increase in lung p52(PAI-1) mRNA content was evident in hyperoxia-treated rats at 24 and 50 h, respectively. All of this increase occurred in the defined 3.2-kb species of rat p52(PAI-1) mRNA. Actin mRNA levels increased three- to sevenfold as a function of hyperoxic stress, whereas catalase and glyceraldehyde-3-phosphate dehydrogenase mRNA abundance was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Involvement of the RNA Polymerase α-Subunit C-Terminal Domain in LuxR-Dependent Activation of the Vibrio fischeri Luminescence Genes

1999 ◽  
Vol 181 (15) ◽  
pp. 4704-4707 ◽  
Author(s):  
Ann M. Stevens ◽  
Nobuyuki Fujita ◽  
Akira Ishihama ◽  
E. P. Greenberg
Keyword(s):  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Vibrio Fischeri ◽  
In Vivo Studies ◽  
Wild Type ◽  
Α Subunit ◽  
Subunit C ◽  
Terminal Domain

ABSTRACT LuxR is a ς70 RNA polymerase (RNAP)-dependent transcriptional activator that controls expression of the Vibrio fischeri lux operon in response to an acylhomoserine lactone-cell density signal. We have investigated whether the α-subunit C-terminal domain (αCTD) of RNAP is required for LuxR activity. A purified signal-independent, LuxR C-terminal domain-containing polypeptide (LuxRΔN) was used to study the activation of transcription from theluxI promoter in vitro. Initiation of luxoperon transcription was observed in the presence of LuxRΔN and wild-type RNAP but not in the presence of LuxRΔN and RNAPs with truncated αCTDs. We also studied the in vivo role of the RNAP αCTD in activation of lux transcription in Escherichia coli. This enabled a comparison of results obtained with full-length LuxR to those obtained with LuxRΔN. These in vivo studies indicated that both LuxR and LuxRΔN require the RNAP αCTD for activity. The results of DNase I protection studies showed that LuxRΔN-RNAP complexes can bind and protect the luxIpromoter, but with less efficacy when the αCTD is truncated in comparison to the wild type. Thus, both in vitro and in vivo experiments demonstrated that LuxR-dependent transcriptional activation of the lux operon involves the RNAP αCTD and suggest that αCTD-LuxR interactions may play a role in recruitment of RNAP to theluxI promoter.

scholarly journals The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

1999 ◽  
Vol 10 (7) ◽  
pp. 2119-2129 ◽  
Author(s):  
Marcus P. Cooper ◽  
Adayabalam S. Balajee ◽  
Vilhelm A. Bohr
Keyword(s):  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Binding Domain ◽  
In Vivo Studies ◽  
C Terminus ◽  
Nucleotide Excision ◽  
N Terminus

The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase δ. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.

scholarly journals Bacterial O-GlcNAcase genes abundance decreases in ulcerative colitis patients and its administration ameliorates colitis in mice

Gut ◽  
2020 ◽  
pp. gutjnl-2020-322468
Author(s):  
Xiaolong He ◽  
Jie Gao ◽  
Liang Peng ◽  
Tongtong Hu ◽  
Yu Wan ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Gut Microbiota ◽  
Catalytic Domain ◽  
Host Cells ◽  
In Vivo Studies ◽  
Post Translational Modification ◽  
Human Gut ◽  
Colonic Inflammation

ObjectiveO-linked N-acetylglucosaminylation (O-GlcNAcylation), controlled by O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), is an important post-translational modification of eukaryotic proteins and plays an essential role in regulating gut inflammation. Gut microbiota encode various enzymes involved in O-GlcNAcylation. However, the characteristics, abundance and function of these enzymes are unknown.DesignWe first investigated the structure and taxonomic distribution of bacterial OGAs and OGTs. Then, we performed metagenomic analysis to explore the OGA genes abundance in health samples and different diseases. Finally, we employed in vitro and in vivo experiments to determine the effects and mechanisms of bacterial OGAs to hydrolyse O-GlcNAcylated proteins in host cells and suppress inflammatory response in the gut.ResultsWe found OGAs, instead of OGTs, are enriched in Bacteroidetes and Firmicutes, the major bacterial divisions in the human gut. Most bacterial OGAs are secreted enzymes with the same conserved catalytic domain as human OGAs. A pooled analysis on 1999 metagenomic samples encompassed six diseases revealed that bacterial OGA genes were conserved in healthy human gut with high abundance, and reduced exclusively in ulcerative colitis. In vitro studies showed that bacterial OGAs could hydrolyse O-GlcNAcylated proteins in host cells, including O-GlcNAcylated NF-κB-p65 subunit, which is important for activating NF-κB signalling. In vivo studies demonstrated that gut bacteria-derived OGAs could protect mice from chemically induced colonic inflammation through hydrolysing O-GlcNAcylated proteins.ConclusionOur results reveal a previously unrecognised enzymatic activity by which gut microbiota influence intestinal physiology and highlight bacterial OGAs as a promising therapeutic strategy in colonic inflammation.

The estrogen-regulated lncRNA H19/miR-216a-5p axis alters stromal cell invasion and migration via ACTA2 in endometriosis

2019 ◽  
Vol 25 (9) ◽  
pp. 550-561 ◽  
Author(s):  
Zhen Xu ◽  
Liping Zhang ◽  
Qian Yu ◽  
Yanan Zhang ◽  
Lei Yan ◽  
...  
Keyword(s):  
Binding Sites ◽  
Stromal Cell ◽  
Cultured Cells ◽  
Fibrous Tissue ◽  
In Vivo Studies ◽  
Invasion And Migration ◽  
Infertile Women ◽  
And Migration

Abstract Fibrotic tissue may contribute to the origin of some endometriosis-related symptoms, such as chronic pelvic pain and infertility. Alterations in the H19/miR-216a-5p/ACTA2 pathway may mediate the regulation of eutopic endometrial stromal cell (euESC) invasion and migration and may represent a potential mechanism underlying fibrous tissue formation or fibrosis in women with endometriosis. In this study, we aimed to determine the expression of H19 and ACTA2 in endometrial tissues of women with endometriosis. Two groups of 23 infertile women with endometriosis and 23 matched infertile women without endometriosis were investigated. Primary cultured cells of endometrial tissues were analyzed using RT-PCR and western blotting (WB) to determine expression of H19 and ACTA2. 5-Ethyl-2′-deoxyuridine, CCK8 and Transwell assays were used to study the functions of H19 and ACTA2. Human embryonic kidney 293 cells were used for luciferase assays to study miR-216a-5p binding sites with H19 and ACTA2. We found that H19 and ACTA2 levels were significantly higher in endometriosis euESCs than in control euESCs (P < 0.05) and were positively correlated in endometriosis euESCs. Luciferase assays indicated that H19 regulates ACTA2 expression via competition for inhibitory miR-216a-5p binding sites. Our results indicate that alterations in the estrogen/H19/miR-216a-5p/ACTA2 pathway regulated endometriosis euESC invasion and migration. Downregulation of H19 or ACTA2 inhibited endometriosis euESC invasion and migration; however, estrogen promoted endometriosis euESC invasion and migration via H19. The main limitation of our study was that experiments were conducted in vitro and further in vivo studies are required in the future. However, our study showed that primary cultured cells represented endometriosis cells more clearly than cell lines.

Isoprenylation of rab proteins on structurally distinct cysteine motifs

1992 ◽  
Vol 102 (4) ◽  
pp. 857-865 ◽  
Author(s):  
M. Peter ◽  
P. Chavrier ◽  
E.A. Nigg ◽  
M. Zerial
Keyword(s):  
Present Report ◽  
Endocytic Pathway ◽  
In Vivo Studies ◽  
Rab Proteins ◽  
Post Translational Modification ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nuclear Lamins ◽  
Cysteine Motifs

rab proteins are low molecular weight GTP-binding proteins highly related to Ypt1p and Sec4p, which are involved in the control of secretion in yeast Saccharomyces cerevisiae. Morphological and biochemical studies have shown that rab proteins are membrane associated and are localized to specific subcompartments along the exocytic and endocytic pathway. Membrane association requires the presence of C-terminal cysteine residues. The present report indicates that the structurally distinct cysteine motifs of rab proteins are subjected to isoprenylation both in vitro and in vivo. Studies on deletion mutants suggest that an intact C-terminal end is required for the association of rab proteins with the membrane and is necessary for the post-translational modification. Finally, we show that the isoprenoid transferase which modifies rab termini is different from the enzyme which farnesylates nuclear lamins and ras proteins in vitro.

Pre-clinical Validation of Mito-targeted Nano-engineered Flavonoids Isolated From Selaginella bryopteris (Sanjeevani) As A Novel Cancer Prevention Strategy

2019 ◽  
Vol 18 (13) ◽  
pp. 1860-1874 ◽  
Author(s):  
Arpit Bhargava ◽  
Neelam Pathak ◽  
Sriram Seshadri ◽  
Neha Bunkar ◽  
Dinesh K. Mishra ◽  
...  
Keyword(s):  
Oral Administration ◽  
Cultured Cells ◽  
Plant Secondary Metabolites ◽  
Human Tumor ◽  
Inhibitory Effect ◽  
In Vivo Studies ◽  
Clinical Validation ◽  
Selaginella Bryopteris

Background: Novel bioactive plant secondary metabolites, including flavonoids, offer a spectrum of chemo-protective responses against a range of human tumor models. However, the clinical translation of these promising anti-cancer agents has been hindered largely by their poor solubility, rapid metabolism, or a combination of both, ultimately resulting in poor bioavailability upon oral administration. Objective: To circumvent the challenges associated with herbal drug development and for effective integration into clinical setting, nano-engineering is one of the emerging pragmatic strategies which has promise to deliver therapeutic concentrations of bio-actives upon oral administration. Method: We assessed the nano-encapsulated flavonoid-rich fraction isolated from a traditional Indian herb Selaginella bryopteris (Sanjeevani) (NP.SB). Both in vitro and in vivo studies were performed to evidence the epigenetic protection mechanisms of NP.SB through a mitochondrial-targeted pre-clinical validation strategy. Results: The mito-protective activity of NP.SB revealed a dose-dependent effect when tested in GC-1 spg (mouse spermatogonial epithelial) and B/CMBA.Ov (mouse ovarian epithelial) following exposure to Nsuccinimidyl N-methylcarbamate, a potential human carcinogen. Smaller size, rapid internalization, faster mobility and site specific delivery conferred significant cancer protection in cultured cells. Notably, this encapsulated flavonoid supplementation; prevented emergence of neoplastic daughter clones from senescent mother phenotypes in pro-oxidant treated GC-1 spg and B/CMBA.Ov cells by selective abrogation of mitochondrial oxidative stress-induced aberrant epigenetic modifications. In vivo studies using a diethylnitrosamine and 2- acetylaminofluorene mouse model demonstrated that NP.SB has a significant inhibitory effect on tumor growth which clearly substantiated our in vitro findings. Conclusion: Anti-carcinogenic property in conjunction with low toxicity of NP.SB, underscores the translational significance of dietary flavonoids as cancer-protective agents for preferential application in clinical settings.

scholarly journals Protease Inhibitory Effect of Natural Polyphenolic Compounds on SARS-CoV-2: An In Silico Study

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4604
Author(s):  
Rajveer Singh ◽  
Anupam Gautam ◽  
Shivani Chandel ◽  
Arijit Ghosh ◽  
Dhritiman Dey ◽  
...  
Keyword(s):  
In Silico ◽  
Inhibitory Effect ◽  
Polyphenolic Compounds ◽  
Host Protein ◽  
In Vivo Studies ◽  
Pharmacokinetic Parameters ◽  
Post Translational Modification ◽  
In Silico Studies

The current pandemic, caused by SARS-CoV-2 virus, is a severe challenge for human health and the world economy. There is an urgent need for development of drugs that can manage this pandemic, as it has already infected 19 million people and led to the death of around 711,277 people worldwide. At this time, in-silico studies are providing lots of preliminary data about potential drugs, which can be a great help in further in-vitro and in-vivo studies. Here, we have selected three polyphenolic compounds, mangiferin, glucogallin, and phlorizin. These compounds are isolated from different natural sources but share structural similarities and have been reported for their antiviral activity. The objective of this study is to analyze and predict the anti-protease activity of these compounds on SARS-CoV-2main protease (Mpro) and TMPRSS2 protein. Both the viral protein and the host protein play an important role in the viral life cycle, such as post-translational modification and viral spike protein priming. This study has been performed by molecular docking of the compounds using PyRx with AutoDock Vina on the two aforementioned targets chosen for this study, i.e., SARS-CoV-2 Mpro and TMPRSS2. The compounds showed good binding affinity and are further analyzed by (Molecular dynamic) MD and Molecular Mechanics Poisson-Boltzmann Surface Area MM-PBSA study. The MD-simulation study has predicted that these natural compounds will have a great impact on the stabilization of the binding cavity of the Mpro of SARS-CoV-2. The predicted pharmacokinetic parameters also show that these compounds are expected to have good solubility and absorption properties. Further predictions for these compounds also showed no involvement in drug-drug interaction and no toxicity.

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