Apoptosis is an important mechanism of cell demise in multicellular organisms and Cl(-) transport has an important role in the progression of the apoptotic volume decrease (AVD).
DIDS (4,4′-Diisothiocyanatostilbene-2,2′-disulfonate) is one of the most commonly used Cl(-) transport inhibitors that eliminates or reduces different apoptotic hallmarks such as AVD, caspase-3 activity and DNA fragmentation.
DIDS is also a protein crosslinker that alkylates either amino or thiol groups.
Since caspases are thiol proteases, our aim was to study whether DIDS could directly inhibit the activity of these proteases.
Here, we show that caspase activity induced by 4 h incubation with staurosporine was inhibited by DIDS in HeLa cells that were maintained in the absence of serum for 24 h.
Interestingly, the caspase-inhibitory effect of DIDS is downstream to the inhibition of cytochrome c release, suggesting that DIDS might be also acting at the apoptosome.
Moreover, DIDS was able to inhibit capase-3, -9, and -8 activities in cell lysates, implying that DIDS can react with and directly block caspases.
Our data suggest that antiapoptotic activity of DIDS involves not only inhibition of the voltage-dependent anion channel (VDAC) at the mitochondria and Cl(-) channels at the plasma membrane, but also a third mechanism based on the direct inhibition of caspases.
Development and validation of a simple and isocratic reversed-phase HPLC method for the determination of rilpivirine from tablets, nanoparticles and HeLa cell lysates.
In the present investigation, a simple and isocratic HPLC-UV method was developed and validated for determination of rilpivirine (RPV) from dosage forms (tablets and nanoparticles) and biological matrices like HeLa cell lysates.
The separation and analysis of RPV was carried out under isocratic conditions using (a) a Gemini reversed-phase C18 column (5 µm; 4.6 × 150 mm) maintained at 35°C, (b) a mobile phase consisting of a mixture of acetonitrile and 25 m m potassium dihydrogen phosphate (in the ratio 50:50 v/v) at a flow rate of 0.6 mL/min and (c) atazanavir as an internal standard.
The total run time was 17 min and the analysis of RPV and internal standard was carried out at 290 nm.
The method was found to be linear (r(2) value>> 0.998), specific, accurate and precise over the concentration range of 0.025-2 µg/mL. The lower limit of quantification was 0.025 µg/mL, the limit of detection was 0.008 µg/mL and the recovery of RPV was >90%.
The stability of the RPV analytical method was confirmed at various conditions such as room temperature (24 h), -20°C (7 days), three freeze-thaw cycles and storage in an autosampler (4°C for 48 h).
The method was successfully applied for the determination of RPV from conventional dosage forms like tablets, from polymeric nanoparticles and from biological matrices like HeLa cell lysates.
Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.
Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans.
The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates.
Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations.
Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway.
CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide.
In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain.
An experimental protocol for the fractionation and 2DE separation of HeLa and A-253 cell lysates suitable for the identification of the individual antigenic proteome in Sjögren’s syndrome.
Sjögren’s syndrome (SS) is an autoimmune disease affecting exocrine glands, especially the salivary and lacrimal glands. Although most of the SS patients’ sera have autoantibodies that can target a variety of antigens, it is not clear what determines which proteins will become autoantigens.
The muscarinic receptor M3, an integral plasma membrane protein, has been proposed as a possible autoantigen in SS, and is endogenous in HeLa cells. The aim of this study was to develop a method that is able to separate and identify antigens recognised by sera from SS patients using lysates of HeLa and A-253 cells in 2D Western Blot (2DWB). The HeLa and A-253 cell lysates were fractionated in soluble and membrane-bound proteins, and the membrane-bound proteins were enriched for integral proteins.
The fractions were tested using WB, confirming the presence of the main cell compartments. The rehydration solution containing ASB-14 performed better than the others in all three steps (active rehydration, focus and transfer), and efficiently separated the muscarinic receptor M3.
The M3 receptor was also detected in lysates from A-253 cells. The presence of this receptor in this cell line has not been proven earlier.
This work develops a suitable protocol to perform a mapping of the autoantibodies present in the sera of single SS patients, using lysates from epithelial cell lines that represent the main cell compartments as an antigen source.
It is our future aim to use this protocol to perform a mapping of the antibodies present in the sera of individual SS patients.
Alphacoronavirus transmissible gastroenteritis virus nsp1 protein suppresses protein translation in mammalian cells and in cell-free HeLa cell extracts but not in rabbit reticulocyte lysate.
The nsp1 protein of transmissible gastroenteritis virus (TGEV), an alphacoronavirus, efficiently suppressed protein synthesis in mammalian cells.
Unlike the nsp1 protein of severe acute respiratory syndrome coronavirus, a betacoronavirus, the TGEV nsp1 protein was unable to bind 40S ribosomal subunits or promote host mRNA degradation.
TGEV nsp1 also suppressed protein translation in cell-free HeLa cell extract; however, it did not affect translation in rabbit reticulocyte lysate (RRL).
Our data suggested that HeLa cell extracts and cultured host cells, but not RRL, contain a host factor(s) that is essential for TGEV nsp1-induced translational suppression.
G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates.
Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes.
We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs.
We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures.
The apparent K(d) values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 +/- 6 and 77 +/- 6 Pm, respectively, as measured by gel mobility shift assay.
In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution.
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Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells.
This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures.