Mechanism Of Cell Lysis Research Articles

Characterization of the LysP2110-HolP2110 Lysis System in Ralstonia solanacearum Phage P2110.

Ralstonia solanacearum, a pathogen causing widespread bacterial wilt disease in numerous crops, currently lacks an optimal control agent. Given the limitations of traditional chemical control methods, including the risk of engendering drug-resistant strains and environmental harm, there is a dire need for sustainable alternatives. One alternative is lysin proteins that selectively lyse bacteria without contributing to resistance development. This work explored the biocontrol potential of the LysP2110-HolP2110 system of Ralstonia solanacearum phage P2110. Bioinformatics analyses pinpointed this system as the primary phage-mediated host cell lysis mechanism. Our data suggest that LysP2110, a member of the Muraidase superfamily, requires HolP2110 for efficient bacterial lysis, presumably via translocation across the bacterial membrane. LysP2110 also exhibits broad-spectrum antibacterial activity in the presence of the outer membrane permeabilizer EDTA. Additionally, we identified HolP2110 as a distinct holin structure unique to the Ralstonia phages, underscoring its crucial role in controlling bacterial lysis through its effect on bacterial ATP levels. These findings provide valuable insights into the function of the LysP2110-HolP2110 lysis system and establish LysP2110 as a promising antimicrobial agent for biocontrol applications. This study underpins the potential of these findings in developing effective and environment-friendly biocontrol strategies against bacterial wilt and other crop diseases.

Anticancer Activity of Nano-formulated Orlistat-Dopamine Conjugates Through Self-Assembly.

Orlistat, an FDA-approved fatty acid inhibitor for obesity treatment, demonstrates certain low and greatly varied anticancer abilities. In a previous study, we revealed a synergistic effect between orlistat and dopamine in cancer treatment. Here, orlistat-dopamine conjugates (ODCs) with defined chemical structures were synthesized. The ODC by design underwent polymerization and self-assembly in the presence of oxygen to form nano-sized particles (Nano-ODCs) spontaneously. The resulted Nano-ODCs of partial crystalline structures demonstrated good water dispersion to form stable Nano-ODC suspensions. Because of the bioadhesive property of the catechol moieties, once administered, Nano-ODCs were quickly accumulated on cell surfaces and efficiently uptaken by cancer cells. In the cytoplasm, Nano-ODC experienced biphasic dissolution followed by spontaneous hydrolysis to release intact orlistat and dopamine. Besides elevated levels of intracellular reactive oxygen species (ROS), the co-localized dopamine also induced mitochondrial dysfunctions through monoamine oxidases (MAOs)-catalyzed dopamine oxidation. The strong synergistic effects between orlistat and dopamine determined a good cytotoxicity activity and a unique cell lysis mechanism, explaining the distinguished activity of Nano-ODC to drug-sensitive and -resistant cancer cells. This new technology-enabled orlistat repurposing will contribute to overcoming drug resistance and the improvement of cancer chemotherapy.

A Simple and Efficient Mechanical Cell Disruption Method Using Glass Beads to Extract β-Glucans from Spent Brewer’s Yeast

β-glucan extraction from spent brewer’s yeast is a long process that starts with the lysis of yeast cells, this step lasting up to 36 h and can be disadvantageous when working on a small scale. In this study, a rapid cell rupture method was selected for the lysis of spent brewer’s yeast to obtain β-glucans. Optimal parameters were determined for the lysis of a cellular suspension of spent brewer’s yeast by vortexing with glass beads. Thus, parameters such as the number of 10 min vortex cycles from 1 to 3, the concentration of cell suspension (5, 10, and 15%), and the ratio of yeast/glass beads (1:1, 1:2, and 1:3) were varied in a Box-Behnken design. A cell lysis mechanism using glass beads allows the cell to rupture and permits the removal of intracellular content. An increase in yeast suspension concentration decreased the disruption efficiency, while a proportional increase was observed with the yeast/glass beads ratio and the increasing number of vortexing cycles. The optimal parameters for cell lysis were found to be a cell suspension concentration of 5%, a ratio of yeast/glass beads of 1:2, and a vortexing cycle of 3, with a disruption efficiency of 99.8%. The β-glucan fraction extracted from the optimal sample showed characteristic absorption bands at 1370.77 and 1153.92 cm−1, the content of β-glucan being 78.53%.

Phage-Mediated Explosive Cell Lysis Induces the Formation of a Different Type of O-IMV in Shewanella vesiculosa M7T.

Shewanella vesiculosa M7T is a cold-adapted Antarctic bacterium that has a great capacity to secrete membrane vesicles (MVs), making it a potentially excellent model for studying the vesiculation process. S. vesiculosa M7T undergoes a blebbing mechanism to produce different types of MVs, including outer membrane vesicles and outer-inner membrane vesicles (O-IMVs). More recently, other mechanisms have been considered that could lead to the formation of O-IMVs derived from prophage-mediated explosive cell lysis in other bacteria, but it is not clear if they are of the same type. The bacterial growth phase could also have a great impact on the type of MVs, although there are few studies on the subject. In this study, we used high-resolution flow cytometry, transmission electron microscopy, and cryo-electron microscopy (Cryo-EM) analysis to determine the amount and types of MVs S. vesiculosa M7T secreted during different growth phases. We show that MV secretion increases during the transition from the late exponential to the stationary phase. Moreover, prophage-mediated explosive cell lysis is activated in S. vesiculosa M7T, increasing the heterogeneity of both single- and double-layer MVs. The sequenced DNA fragments from the MVs covered the entire genome, confirming this explosive cell lysis mechanism. A different structure and biogenesis mechanisms for the explosive cell lysis-derived double-layered MVs was observed, and we propose to name them explosive O-IMVs, distinguishing them from the blebbing O-IMVs; their separation is a first step to elucidate their different functions. In our study, we used for the first time sorting by flow cytometry and Cryo-EM analyses to isolate bacterial MVs based on their nucleic acid content. Further improvements and implementation of bacterial MV separation techniques is essential to develop more in-depth knowledge of MVs.

Mature viral cathepsin is required for release of viral occlusion bodies from Autographa californica multiple nucleopolyhedrovirus-infected cells

Baculovirus-infected larvae release progeny viral occlusion bodies (OBs) to enable cyclical virus transmission to new hosts. The alphabaculovirus chitinase and cathepsin enzymes cause terminal liquefaction of host insect cadavers, aiding OB dispersal. The mechanism of cell lysis required to release the OBs is unclear but here we show Autographa californica multiple nucleopolyhedrovirus cathepsin protease activity is required for efficient release of the host tissue-degrading chitinase and cathepsin enzymes and critical for release of progeny OBs from virus-infected cells. Comparisons between viruses containing or lacking cathepsin indicate that cathepsin was necessary for OB release into cultured cell media or hemolymph of insects. In addition, pharmacological inhibition of cysteine protease activity in cells during infection blocked maturation of active cathepsin and OB release from infected cells. Together, these results suggest an important link between baculovirus-induced cell lysis, the concomitant maturation of cathepsin, and cellular release of chitinase, cathepsin and progeny OBs from cells.

Antimicrobial Hydrogels: Promising Soft Biomaterials

AbstractHydrogel is a three‐dimensional, hydrophilic network of cross‐linked monomer or polymer units. A modified version of the hydrogel which exhibits antimicrobial properties is known as antimicrobial hydrogel. Antimicrobial properties of the antimicrobial hydrogel are due to the inheritance of antimicrobial monomers/polymers or due to the installation by the incorporation of antimicrobial agents such as a metal nanoparticle, metal oxide, antibiotics, and any other synthetic drug molecule. More attention has been taken towards antimicrobial hydrogel as the pharmaceutical and biomedical field facing the problem of drug resistance. Antimicrobial hydrogel provides a suitable alternative for antibiotic drugs and also successful to replace many antibiotic drugs which cause a serious side effect on the human body. Antimicrobial hydrogel targets the cell membrane of the microorganism and destroys them. Due to this unique type of cell lysis mechanism, microbes are rarely able to develop resistance towards antimicrobial hydrogel. The hydrogel is porous, highly flexible, and holds a particular amount of water. These properties impart a moist and living body tissue‐like environment and hence make it a suitable biomaterial to use in biomedical applications. The present article focused on the recent development of bio‐based antimicrobial hydrogels and their applications in drug delivery, wound healing and dressing, tissue engineering, and agriculture.

Conformational Differences Are Observed for the Active and Inactive Forms of Pinholin S21 Using DEER Spectroscopy.

Bacteriophages have evolved with an efficient host cell lysis mechanism to terminate the infection cycle and release the new progeny virions at the optimum time, allowing adaptation with the changing host and environment. Among the lytic proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time known as "holin triggering". Pinholin S21 is a prototype holin of phage Φ21 which makes many nanoscale holes and destroys the proton motive force, which in turn activates the signal anchor release (SAR) endolysin system to degrade the peptidoglycan layer of the host cell and destruction of the outer membrane by the spanin complex. Like many others, phage Φ21 has two holin proteins: active pinholin and antipinholin. The antipinholin form differs only by three extra amino acids at the N-terminus; however, it has a different structural topology and conformation with respect to the membrane. Predefined combinations of active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously, the dynamics and topology of active pinholin and antipinholin were investigated (Ahammad et al. JPCB 2019, 2020) using continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. However, detailed structural studies and direct comparison of these two forms of pinholin S21 are absent in the literature. In this study, the structural topology and conformations of active pinholin (S2168) and inactive antipinholin (S2168IRS) in DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) proteoliposomes were investigated using the four-pulse double electron-electron resonance (DEER) EPR spectroscopic technique to measure distances between transmembrane domains 1 and 2 (TMD1 and TMD2). Five sets of interlabel distances were measured via DEER spectroscopy for both the active and inactive forms of pinholin S21. Structural models of the active pinholin and inactive antipinholin forms in DMPC proteoliposomes were obtained using the experimental DEER distances coupled with the simulated annealing software package Xplor-NIH. TMD2 of S2168 remains in the lipid bilayer, and TMD1 is partially externalized from the bilayer with some residues located on the surface. However, both TMDs remain incorporated in the lipid bilayer for the inactive S2168IRS form. This study demonstrates, for the first time, clear structural topology and conformational differences between the two forms of pinholin S21. This work will pave the way for further studies of other holin systems using the DEER spectroscopic technique and will give structural insight into these biological clocks in molecular detail.

Time-Resolved Single-Cell Lysis Kinetics of Metal Nanoparticle-Infused Biopolymer Matrices

Bacterial infections continue to be a growing concern in healthcare facilities due to the increasing number of multi-drug resistant bacteria. The extensive use of antibiotics has weakened the potency and effectiveness of these drugs, consequently promoting bacterial infections and extending healing time. Therefore, there is an immediate demand to find an alternate method to combat the spread of bacterial infections. Metal nanoparticles (MNP) have proven to be an effective antibacterial agent against antibiotic-resistant bacteria, and when incorporated into polymeric substrates, have exhibited high toxicity towards bacterial cells. Current research proposes that MNP interact with the bacterial cell wall, undergo redox processes to form ions, and the ions diffuse into the cell resulting in disruption of membrane-bound proteins. This results in the inhibition of cell replication or cell lysis. However, existing methods of antimicrobial susceptibility testing, such as broth dilution assays and disk diffusion, fail to provide a greater understanding of the time scale required for the single cell lysis process to occur. More specifically, the conventional methods have substrate and agent limitations, as well as assume homogeneity in the bacterial population, therefore making the techniques an ineffective method for probing the cell lysis mechanism of MNP-infused biopolymer matrices. To further investigate the mechanistic action of metal nanocomposites, this research utilizes epi-fluorescence optical tweezers for antibacterial characterization. More specifically, a single E. coli cell was optically trapped, non-covalently docked to MNP-infused alginate matrix, and the cell viability kinetics were monitored in situ. Initial results showed that antibacterial activity of Cu and Ag functionalized nanocomposites was related to the metal concentration and size of colloidal NP. Ag incorporated polymers exhibited greater antibacterial effectiveness when compared to Cu potentially due to the role of Cu in cellular respiration processes. Results further indicate that the size of the MNP does impact the rate of single cell lysis, which may be related to the redox kinetics at the cell/MNP interface.

A Mechanistic Perspective on Process-Induced Changes in Glucosinolate Content in Brassica Vegetables: A Review

Brassica vegetables are consumed mostly after processing, which is expected to give beneficial effects on the vegetable properties, such as improved palatability and bioavailability of nutrients, or shelf life extension. But processing also results to various changes in the content of health promoting phytochemicals like glucosinolates. This paper reviews the effects of processing on the glucosinolates content by using a mechanism approach underlying processing method employed. Cultural differences between Eastern and Western preparation practices and their possible effect on glucosinolate retention are highlighted. Boiling and blanching considerably reduce the glucosinolate content mainly due to mechanisms of cell lysis, diffusion, and leaching, and partly due to thermal and enzymatic degradation. Steaming, microwave processing, and stir frying either retain or slightly reduce the glucosinolates content due to low degrees of leaching; moreover, these methods seem to enhance extractability of glucosinolates from the plant tissue. Fermentation reduces the glucosinolate content considerably, but the underlying mechanisms are not yet studied in detail. Studying the changes of glucosinolates during processing by a mechanistic approach is shown to be valuable to understand the impact of processing and to optimize processing conditions for health benefits of these compounds.

Phosphoinositide-mediated oligomerization of a defensin induces cell lysis

Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique 'cationic grip' configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001.

Peptide Self-Assembly on Cell Membranes to Induce Cell Lysis

Self-assembling into aggregates with defined structures is a common phenomenon for many peptides at high concentrations. In this study, we found that when PTP-7b (FLGALFKALSHLL), a concentration-dependent self-assembling peptide, bound to tissue cells and accumulated on cell surfaces, it migrated and self-assembled into exosome-like aggregates at certain locations on the cell membranes. Studies using confocal microscopy and scanning electron microscopy revealed that peptide PTP-7b induced cell tissue damage through a new cell lysis mechanism that involved peptide self-assembly on cell surfaces, extracting lipids from cell membranes, and transporting peptides into the cytoplasm. Peptide self-assembly attributed greatly to peptide-cell interactions and thus the biological activity of a peptide. Because peptide self-assembly was a slow process, PTP-7b-induced cell lysis showed a biphasic behavior: a gradual viability decrease was followed by a rapid decline. These results suggest that peptide self-assembly could be equally as important as charge and secondary structure of a peptide in determining the anticancer and antibacterial activities of therapeutic peptides.

Human Adenovirus Type 5 Induces Cell Lysis through Autophagy and Autophagy-Triggered Caspase Activity

Oncolytic adenoviruses, such as Delta-24-RGD, are promising therapies for patients with brain tumor. Clinical trials have shown that the potency of these cancer-selective adenoviruses should be increased to optimize therapeutic efficacy. One potential strategy is to increase the efficiency of adenovirus-induced cell lysis, a mechanism that has not been clearly described. In this study, for the first time, we report that autophagy plays a role in adenovirus-induced cell lysis. At the late stage after adenovirus infection, numerous autophagic vacuoles accompany the disruption of cellular structure, leading to cell lysis. The virus induces a complete autophagic process from autophagosome initiation to its turnover through fusion with the lysosome although the formation of the autophagosome is sufficient for virally induced cell lysis. Importantly, downmodulation of autophagy genes (ATG5 or ATG10) rescues the infected cells from being lysed by the virus. Moreover, autophagy triggers caspase activity via the extrinsic FADD/caspase 8 pathway, which also contributes to adenovirus-mediated cell lysis. Therefore, our study implicates autophagy and caspase activation as part of the mechanism for cell lysis induced by adenovirus and suggests that manipulation of the process is a potential strategy to optimize clinical efficacy of oncolytic adenoviruses.

Various physico-chemical stress factors cause prophage induction in Nitrosospira multiformis 25196- an ammonia oxidizing bacteria

Bacteriophages are virsues that infect bacteria and contribute significant changes in the overall bacterial community. Prophages are formed when temperate bacteriophages integrate their DNA into the bacterial chromosome during the lysogenic cycle of the phage infection to bacteria. The prophage (phage DNA integrated into bacterial genome) on the bacterial genome remains dormant, but can cause cell lysis under certain environmental conditions. This research examined the effect of various environmental stress factors on the ammonia oxidation and prophage induction in a model ammonia oxidizing bacteria Nitrosospira multiformis ATCC 25196. The factors included in the study were pH, temperature, organic carbon (COD), the presence of heavy metal in the form of chromium (VI) and the toxicity as potassium cyanide (KCN). The selected environmental factors are commonly encountered in wastewater treatment processes, where ammonia oxidizing bacteria play a pivotal role of converting ammonia into nitrite. All the factors could induce prophage from N. multiformis demonstrating that cell lysis due to prophage induction could be an important mechanism contributing to the frequent upset in ammonia oxidation efficiency in full scale treatment plants. Among the stress factors considered, pH in the acidic range was the most detrimental to the nitrification efficiency by N. multiformis. The number of virus like particles (VLPs) increased by 2.3E+10 at pH 5 in 5 h under acidic pH conditions. The corresponding increases in VLPs at pH values of 7 and 8 were 9.67E+9 and 1.57E+10 in 5 h respectively. Cell lysis due to stress resulting in phage induction seemed the primary reason for deteriorated ammonia oxidation by N. multiformis at lower concentrations of Cr (VI) and potassium cyanide. However, direct killing of N. multiformis due to the binding of Cr (VI) and potassium cyanide with cell protein as demonstrated in the literature at higher concentrations of these toxic compounds was the primary mechanism of cell lysis of N. multiformis. Organics represented by the chemical oxygen demand (COD) did not have any effect on the phage induction in N. multiformis. This AOB remained dormant at low temperature (4 °C) without any phage induction. Significant decrease in the number of live N. multiformis cells with a corresponding increase in the number of VLPs was recorded when the temperature was increased to 35 °C. Death of N. multiformis at 45 °C was attributed to the destruction of cell wall rather than to the phage induction.

Rapid β-lactam-induced lysis requires successful assembly of the cell division machinery

Beta-lactam antibiotics inhibit penicillin binding proteins (PBPs) involved in peptidoglycan synthesis. Although inhibition of peptidoglycan biosynthesis is generally thought to induce cell lysis, the pattern and mechanism of cell lysis can vary substantially. Beta-lactams that inhibit FtsI, the only division specific PBP, block cell division and result in growth as filaments. These filaments ultimately lyse through a poorly understood mechanism. Here we find that one such beta-lactam, cephalexin, can, under certain conditions, lead instead to rapid lysis at nascent division sites through a process that requires the complete and ordered assembly of the divisome, the essential machinery involved in cell division. We propose that this assembly process (in which the localization of cell wall hydrolases depends on properly targeted FtsN, which in turn depends on the presence of FtsI) ensures that the biosynthetic machinery to form new septa is in place before the machinery to degrade septated daughter cells is enabled. Beta-lactams that target FtsI subvert this mechanism by inhibiting FtsI without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. One seemingly paradoxical implication of our results is that beta-lactam therapy may be improved by promoting active cell division.

Peptide alarmone signalling triggers an auto‐active bacteriocin necessary for genetic competence

The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the 'death effector'. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm.

Expression of Cytotoxicity-Associated Genes in Marek's Disease Virus—Infected Chickens

Cytotoxic host responses to Marek's disease virus (MDV) have been attributed to both natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). However, the mechanisms of cell lysis initiated by these cytotoxic responses during MDV infection are not well defined. Therefore, the current study was aimed at elucidating the molecular mechanisms of host cytotoxic responses to MDV infection by investigating the expression of genes in the cell lysis pathway involving granzyme A. Genes encoding cytolytic proteins, NK lysin, and granzyme A were upregulated during early stages of infection, whereas the genes encoding major histocompatibility complex (MHC) class I and the DNA repair and apoptosis protein, poly(ADP-ribose) polymerase (PARP), were downregulated. These findings shed more light on the mechanisms of host response to MDV infection in chickens.

Effect of combination inhibition of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGF-R) on ovarian cancer cell lines

13112 Background: Bevacizumab, a VEGF inhibitor, and cetuximab, an EGF-R inhibitor, have shown promising results in the treatment of solid tumors. The combination of these two antibodies and combinations with platinum chemotherapy is of interest for the treatment of ovarian cancer. Our objective is evaluate the mechanism of action and to determine the direct effect on tumor proliferation, the role of antibody dependent cell cytotoxicity (ADCC) with immune effector cells and the outcome in nude mouse xenografts with antibody treatment combinations. Methods: Five ovarian cancer cell lines were studied; SKOV3, SKOV8, OVCAR3, A2780, and CaOV3. VEGF secretion levels were determined for each cell line by standard ELISA. EGF-R, VEGF-R and VEGF surface expression was determined by flow cytometry. Cytotoxicity alamar blue 96-well plate assays were performed and cells were plated and exposed to bevacizumab or cetuximab alone, in combination and with cisplatin. Standard 51chromium release assays were performed to determine if bevacizumab and cetuximab have a role in ADCC when combined with peripheral blood mononuclear cells (PBMC). Results: All ovarian cancer cell lines secreted VEGF ranging from 210 to 1,849 pg/ml. Flow cytometry studies demonstrated varying degrees of expression of VEGF-R and minimal surface expression of VEGF ligand. EGF-R was present on all cell lines. Five day alamar blue cytotoxicity assays using varying concentrations of single agent bevacizumab (0.1–10 μM) or single agent cetuximab (0.01–1.0 μM) had minimal to no cytotoxic effect on ovarian cancer cell lines. The IC50 for cisplatin in these cell lines was obtained and ranged from 1–5 μM and was not altered by the addition of antibodies. In 51chromium release assays, the combination of bevacizumab and cetuximab caused a trend toward increased SKOV3 cell lysis by PBMC. Conclusion: Bevacizumab or cetuximab alone or in combination with each other in vitro had minimal direct cytotoxic effect. ADCC may be a mechanism of cell lysis when these antibodies are combined with effector cells (PBMC) and further studies are underway. In vivo studies with nude mouse SKOV3 xenografts are being performed to evaluate the effect on combination antibody therapy on established tumors. No significant financial relationships to disclose.

Immunomodulatory impact of interferon-alpha in combination with chemoradiation of pancreatic adenocarcinoma (CapRI).

Data from a phase II trial combining chemoradiotherapy with interferon-alpha (IFN-alpha) (CapRI scheme) for adjuvant treatment of pancreatic carcinoma are very encouraging. Eight human ductal pancreatic carcinoma cell lines were treated with the CapRI scheme [5-fluorouracil (5-FU), Cisplatin, IFN-alpha and radiation]. Natural killer (NK) and T cells preincubated with IFN-alpha were tested in cytotoxicity assays against these cell lines and the mechanism of cell lysis was investigated. The induction of the immunoproteasome in tumour cells after IFN-alpha stimulation was analysed by immunoblot and RT-PCR. IFN-alpha activated NK cells and increased their cytotoxicity. This cytotoxicity was mediated as well by Fas-induced apoptosis as by perforin release. Pre-treatment of tumour cells with 5-FU and combinations showed a significant increase in the susceptibility of tumour cells against NK cells. Treatment of tumour cells with IFN-alpha induced a switch to the immunoproteasome and enhanced their vulnerability to T cells. This is the first description of this phenomenon in pancreatic carcinoma cells with implications for their immunogenicity. IFN-alpha activates NK cells against pancreatic carcinoma cells and 5-FU treatment makes tumour cells more susceptible. Furthermore, IFN-alpha induces the immunoproteasome with impact on the immunogenicity of pancreatic carcinoma cells. These mechanisms may be responsible for the improved clinical outcome of CapRI.

Molecular dynamics investigation of the influence of anionic and zwitterionic interfaces on antimicrobial peptides’ structure: Implications for peptide toxicity and activity

Molecular dynamics simulations of three related helical antimicrobial peptides have been carried out in zwitterionic diphosphocholine (DPC) micelles and anionic sodiumdodecylsulfate (SDS) micelles. These systems can be considered as model mammalian and bacterial membrane interfaces, respectively. The goal of this study is to dissect the differences in peptide composition which make the mutant peptides (novispirin-G10 and novispirin-T7) less toxic than the parent peptide ovispirin (OVIS), although all three peptides have highly antibacterial properties. Compared to G10 and T7, OVIS inserts deepest into the DPC micelle. This correlates well with the lesser toxicity of G10 and T7. There is strong evidence which suggests that synergistic binding of hydrophobic residues drives binding of OVIS to the micelle. The helical content of G10 and T7 is reduced in the presence of DPC, and this leads to less amphipathic peptide structures, which bind weakly to the micelle. Simulations in SDS were carried out to compare the influence of membrane electrostatics on peptide structure. All three peptides bound strongly to SDS, and retained helical form. This corresponds well with their equally potent antibacterial properties. Based on the simulations, we argue that secondary structure stability often leads to toxic properties. We also propose that G10 and T7 operate by the carpet mechanism of cell lysis. Toxicity of peptides operating by the carpet mechanism can be attenuated by reducing the peptide helical content. The simulations successfully capture experimental binding states, and the different depths of binding of the three peptides to the two micelles correlate with their antibacterial and toxic properties.

On the mechanism of cell lysis by deformation

In this study, we identify the extent of deformation that causes cell lysis using a simple technique where a drop of cell suspension is compressed by two flat plates. The viability of human prostatic adenocarcinoma PC-3 cells in solutions of various concentrations of NaCl is determined as a function of the gap size between the plates. The viability declines with decreasing gap size in the following order: 700 mM>150 mM>75 mM NaCl. This is considered to be due to the difference in cell size, which is caused by the osmotic volume change before deformation; cell diameter becomes smaller in a solution of higher NaCl concentration, which appears to increase the survival ratio in a given gap size. The deformation-induced decrease in cell viability is correlated with the cell surface strain, which is dependent on the increase in surface area, irrespective of NaCl concentration. In addition, the treatment of cells with cytochalasin D results in the disappearance of cortical actin filaments and a marked drop in the viability, indicating that cell lysis is closely related to the deformation of the cytoskeleton.