Doxorubicin and epirubicin iron-induced generation of free radicals In vitro. A comparative study

1987 ◽  
Vol 7 (8) ◽  
pp. 653-658 ◽  
Author(s):  
Kjell Grankvist ◽  
Roger Henriksson
Keyword(s):  
Oxygen Consumption ◽  
Free Radicals ◽  
Myocardial Injury ◽  
Oxygen Free Radicals ◽  
Oxygen Electrode ◽  
Inhibitory Effect ◽  
Free System ◽  
A Cell ◽  
Strong Inhibitory Effect

To ascertain any differences in myocardial injury exerted by the anthracyclines doxorubicin and epirubicin, their ability to generate oxygen free radicals when mixed with Fe(II) was examined in vitro using an oxygen electrode. 5–250 μg/ml doxorubicin or epirubicin consumed oxygen when mixed with 50 or 100 μmol/1 Fe(II). Addition of 75 μmol/1 cytochrome C showed that of the consumed oxygen, approximately 80% entered the monovalent pathway of oxygen reduction. The strong inhibitory effect of 250 mg/1 catalase indicates that most of the superoxide radicals generated are further reduced to hydrogen peroxide by both anthracyclines. Addition of metal chelators DTPA (100/μmol/1), or DDTC (50 μmol/1) did not affect oxygen consumption, whereas EDTA (100/μmol/1) or desferrioxamine (100 μmol/1) with anthracyclines and Fe(II) rather stimulated oxygen consumption. It is concluded that there are no significant differences in the amount or proportion of generated oxygen free radicals between doxorubicin and epirubicin when mixed with Fe(II) in a cell-free system in vitro. Thus, the ability of the anthracyclines, in conjunction with iron alone, to generate radicals does not explain the differences of the drugs in causing myocardial injury.

scholarly journals Stoichiometry of oxygen consumption in the biosynthesis of isopenicillin from a tripeptide

1982 ◽  
Vol 203 (3) ◽  
pp. 791-793 ◽  
Author(s):  
R L White ◽  
E M M John ◽  
J E Baldwin ◽  
E P Abraham
Keyword(s):  
Oxygen Consumption ◽  
Oxygen Electrode ◽  
Stoichiometric Ratio ◽  
O2 Consumption ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Isopenicillin N

The biosynthesis of isopenicillin N from delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in a cell-free system has been correlated wih O2 consumption by two methods, involving the use of an oxygen-electrode and an n.m.r. spectrometer respectively. The results are consistent with a 1 : 1 stoichiometric ratio for the dioxygen consumed to the isopenicillin N formed.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

scholarly journals Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

2010 ◽  
Vol 17 (5) ◽  
pp. 784-792 ◽  
Author(s):  
R. Zichel ◽  
A. Mimran ◽  
A. Keren ◽  
A. Barnea ◽  
I. Steinberger-Levy ◽  
...  
Keyword(s):  
Expression System ◽  
Free Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Dose ◽  
Botulinum Toxins ◽  
Free System ◽  
Toxin Complex ◽  
A Cell ◽  
Cell Free Expression System

ABSTRACT Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni+ affinity chromatography. Mice immunized with three injections containing 5 μg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 105 against the native toxin complex, which enabled protection against a high-dose toxin challenge (103 to 106 mouse 50% lethal dose [MsLD50]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 105 MsLD50 toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

Antinociceptive properties of shikonin: in vitro and in vivo studies

2016 ◽  
Vol 94 (7) ◽  
pp. 788-796 ◽  
Author(s):  
Bhawana Gupta ◽  
Sabyasachi Chakraborty ◽  
Soumya Saha ◽  
Sunita Gulabsingh Chandel ◽  
Atul Kumar Baranwal ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Inhibitory Effect ◽  
Pain Modulation ◽  
In Vivo Studies ◽  
Channel Modulation ◽  
Pain Models ◽  
A Cell ◽  
Dose Dependent

Shikonin possess a diverse spectrum of pharmacological properties in multiple therapeutic areas. However, the nociceptive effect of shikonin is not largely known. To investigate the antinociceptive potential of shikonin, panel of GPCRs, ion channels, and enzymes involved in pain pathogenesis were studied. To evaluate the translation of shikonin efficacy in vivo, it was tested in 3 established rat pain models. Our study reveals that shikonin has significant inhibitory effect on pan sodium channel/N1E115 and NaV1.7 channel with half maximal inhibitory concentration (IC50) value of 7.6 μmol/L and 6.4 μmol/L, respectively, in a cell-based assay. Shikonin exerted significant dose dependent antinociceptive activity at doses of 0.08%, 0.05%, and 0.02% w/v in pinch pain model. In mechanical hyperalgesia model, dose of 10 and 3 mg/kg (intraperitoneal) produced dose-dependent analgesia and showed 67% and 35% reversal of hyperalgesia respectively at 0.5 h. Following oral administration, it showed 39% reversal at 30 mg/kg dose. When tested in first phase of formalin induced pain, shikonin at 10 mg/kg dose inhibited paw flinching by ∼71%. In all studied preclinical models, analgesic effect was similar or better than standard analgesic drugs. The present study unveils the mechanistic role of shikonin on pain modulation, predominantly via sodium channel modulation, suggesting that shikonin could be developed as a potential pain blocker.

scholarly journals Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

1998 ◽  
Vol 18 (10) ◽  
pp. 5670-5677 ◽  
Author(s):  
Ossama Abu Hatoum ◽  
Shlomit Gross-Mesilaty ◽  
Kristin Breitschopf ◽  
Aviad Hoffman ◽  
Hedva Gonen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
26S Proteasome ◽  
Intact Cells ◽  
Free System ◽  
Ubiquitin System

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

A somatic cell-derived system for studying both early and late mitotic events in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 449-462
Author(s):  
J. Nakagawa ◽  
G.T. Kitten ◽  
E.A. Nigg
Keyword(s):  
Chromatin Condensation ◽  
Nuclear Lamina ◽  
Free System ◽  
Cell Free System ◽  
Protein Substrates ◽  
Nuclear Envelope Breakdown ◽  
Biochemical Fractionation ◽  
A Cell

We describe a cell-free system for studying mitotic reorganization of nuclear structure. The system utilizes soluble extracts prepared from metaphase-arrested somatic chicken cells and supports both the disassembly and subsequent partial reassembly of exogenous nuclei. By fluorescence microscopy, biochemical fractionation, protein phosphorylation assays and electron microscopy, we show that chicken embryonic nuclei incubated in extracts prepared from metaphase-arrested chicken hepatoma cells undergo nuclear envelope breakdown, lamina depolymerization and chromatin condensation. These prophase-like events are strictly dependent on ATP and do not occur when nuclei are incubated in interphase extracts. Compared to interphase extracts, metaphase extracts show increased kinase activities toward a number of nuclear protein substrates, including lamins and histone H1; moreover, they specifically contain four soluble phosphoproteins of Mr 38,000, 75,000, 95,000 and 165,000. Following disassembly of exogenous nuclei in metaphase extracts, telophase-like reassembly of a nuclear lamina and re-formation of nuclear membranes around condensed chromatin can be induced by depletion of ATP from the extract. We anticipate that this reversible cell-free system will contribute to the identification and characterization of factors involved in regulatory and mechanistic aspects of mitosis.

Design, synthesis and antibacterial evaluation of ocotillol derivatives with polycyclic nitrogen-containing groups

2021 ◽  
Author(s):  
Yucheng Cao ◽  
Kaiyi Wang ◽  
Jiali Wang ◽  
Haoran Cheng ◽  
Mengxin Ma ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Multidrug Resistant ◽  
Inhibitory Effect ◽  
Resistant Bacteria ◽  
Design Synthesis ◽  
In Vitro Antibacterial Activity ◽  
Strong Inhibitory Effect ◽  
Hospital Acquired ◽  
Derivatives Of

Aim: With the increasing abuse of antibacterial drugs, multidrug-resistant bacteria have become a burden on human health and the healthcare system. To find alternative compounds effective against hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA), novel derivatives of ocotillol were synthesized. Methods & Results: Ocotillol derivatives with polycyclic nitrogen-containing groups were synthesized and evaluated for in vitro antibacterial activity. Compounds 36–39 exhibited potent antibacterial activity against hospital-acquired MRSA, with MIC = 8–64 μg/ml. Additionally, a combination of compound 37 and the commercially available antibiotic kanamycin showed synergistic inhibitory effects, with a fractional inhibitory concentration index of ≤0.375. Conclusion: Compound 37 has a strong inhibitory effect, and this derivative has potential for use as a pharmacological tool to explore antibacterial mechanisms.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters

1981 ◽  
Vol 1 (7) ◽  
pp. 635-651
Author(s):  
D C Lee ◽  
R G Roeder
Keyword(s):  
Adenovirus Type ◽  
Free System ◽  
Ribonucleic Acids ◽  
Cell Free System ◽  
Cell Extracts ◽  
A Cell ◽  
Adenovirus Type 2

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

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