Disruption Of Cellular Membranes Research Articles

Macrophage-Engineered Vesicles for Therapeutic Delivery and Bidirectional Reprogramming of Immune Cell Polarization.

Macrophages, one of the most important phagocytic cells of the immune system, are highly plastic and are known to exhibit diverse roles under different pathological conditions. The ability to repolarize macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) or vice versa offers a promising therapeutic approach for treating various diseases such as traumatic injury and cancer. Herein, it is demonstrated that macrophage-engineered vesicles (MEVs) generated by disruption of macrophage cellular membranes can be used as nanocarriers capable of reprogramming macrophages and microglia toward either pro- or anti-inflammatory phenotypes. MEVs can be produced at high yields and easily loaded with diagnostic molecules or chemotherapeutics and delivered to both macrophages and cancer cells in vitro and in vivo. Overall, MEVs show promise as potential delivery vehicles for both therapeutics and their ability to controllably modulate macrophage/microglia inflammatory phenotypes.

Antioksidanti iz semena kukuruza i ubrzano starenje

Seed deterioration is expressed as the loss of viability, vigour and quality during ageing. The major causes of seed deterioration refer to free radical-mediated lipid peroxidation, enzyme inactivation or protein degradation, disruption of cellular membranes and damage to genetic integrity. Accelerated ageing (AA) is a simple method for studying the mechanisms of deterioration over short periods. In order to estimate the deterioration rate, seeds of four maize genotypes were exposed to accelerated ageing for different time points (three and six days of AA). In parallel, the same genotypes were evaluated regarding the content of total phenolics, phenolic acids, flavonoids and anthocyanins, as well as regarding total antioxidant capacity. Compared to non-stressed seeds, ageing was evidenced by decreased germination energy (48 .9%), a total number of seedlings (40 .8%) and seedlings growth (70 .0% for root length, 44 .0% and 10 .5% for root and shoot fresh weight, 5 .0% for seed rest fresh weight, 12 .0% and 11 .5% for root and shoot dry weight, 15 .3% for seed rest dry weight, respectively), as well as by an increased number of abnormal seedlings (22 .0%). Correlation analysis revealed that higher content of total phenolics, flavonoids and anthocyanins contributed to a higher radical scavenging activity, which was reflected through a lower level of deterioration rate, both regarding the energy of germination and overall seedling performance.

Aluminium oxide nanoparticles inhibit EPS production, adhesion and biofilm formation by multidrug resistant Acinetobacter baumannii

Acinetobacter baumannii is a biofilm forming multidrug resistant (MDR) pathogen responsible for respiratory tract infections. In this study, aluminium oxide nanoparticles (Al2O3 NPs) were synthesized and characterized by TEM and EDX and shown to be spherical shaped nanoparticles with a diameter < 10 nm. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) for the Al2O3 NPs ranged between 125 and 1,000 µg ml−1. Exposure to NPs caused cellular membrane disruption, indicated by an increase in cellular leakage of the contents. Biofilm inhibition was 11.64 to 70.2%, whereas attachment of bacteria to polystyrene surfaces was reduced to 48.8 to 51.9% in the presence of NPs. Nanoparticles also reduced extracellular polymeric substance production and the biomass of established biofilms. The data revealed the non-toxic nature of Al2O3 NPs up to a concentrations of 120 µg ml−1 in HeLa cell lines. These results demonstrate an effective and safer use of Al2O3 NPs against the MDR A. baumannii by targeting biofilm formation, adhesion and EPS production.

Effect of Silver Nanoparticles on Biofilm Formation and EPS Production of Multidrug-Resistant Klebsiella pneumoniae.

Antibiotic resistance against present antibiotics is rising at an alarming rate with need for discovery of advanced methods to treat infections caused by resistant pathogens. Silver nanoparticles are known to exhibit satisfactory antibacterial and antibiofilm activity against different pathogens. In the present study, the AgNPs were synthesized chemically and characterized by UV-Visible spectroscopy, scanning electron microscopy, and X-ray diffraction. Antibacterial activity against MDR K. pneumoniae strains was evaluated by agar diffusion and broth microdilution assay. Cellular protein leakage was determined by the Bradford assay. The effect of AgNPs on production on extracellular polymeric substances was evaluated. Biofilm formation was assessed by tube method qualitatively and quantitatively by the microtiter plate assay. The cytotoxic potential of AgNPs on HeLa cell lines was also determined. AgNPs exhibited an MIC of 62.5 and 125 μg/ml, while their MBC is 250 and 500 μg/ml. The production of extracellular polymeric substance decreased after AgNP treatment while cellular protein leakage increased due to higher rates of cellular membrane disruption by AgNPs. The percentage biofilm inhibition was evaluated to be 64% for K. pneumoniae strain MF953600 and 86% for MF953599 at AgNP concentration of 100 μg/ml. AgNPs were evaluated to be minimally cytotoxic and safe at concentrations of 15-120 μg/ml. The data evaluated by this study provided evidence of AgNPs being safe antibacterial and antibiofilm compounds against MDR K. pneumoniae.

Protective effect of Lactobacillus casei HY2782 against particulate matter toxicity in human intestinal CCD-18Co cells and Caenorhabditis elegans.

To investigate the preventive effect of Lactobacillus casei HY2782 on toxicity induced by particulate matter (PM, inhalable particles less than 10 μm in diameter) in human intestinal CCD-18Co cells and a model animal Caenorhabditis elegans. L. casei HY2782 treatment prevented PM-induced intestinal cell death via cellular reactive oxygen species production and membrane disruption attenuation. PM significantly decreased the total number of eggs laid and the body bending activity of C. elegans, demonstrating PM toxicity. L. casei HY2782 treatment restored the reproductive toxicity and decline in locomotion activity induced by PM in C. elegans. Overall, L. casei HY2782 attenuated PM toxicity in vitro in cultured intestinal cells and in vivo in the model nematode. Our study provides a potential clue for developing L. casei HY2782 probiotics that attenuate PM-induced cellular and physiological toxicity; however, further in-depth preclinical trials using mammalian animal models and clinical trials are required.

Fluorination effect to intermediate molecular weight polyethylenimine for gene delivery systems.

Fluorinated intermediate molecular weight polyethylenimine (FP2ks) with various fluorination degrees was synthesized by conjugation with heptafluorobutyric anhydride and the fluorination effect for gene delivery systems was examined. FP2ks could condense pDNA, forming compact, positively charged, and nano-sized spherical particles. It was thought that their decreased electrostatic interaction with pDNA would be compensated by hydrophobic interaction. The cytotoxicity of FP2ks was increased with the increase of fluorination degree, probably due to the cellular membrane disruption via hydrophobic interaction with FP2ks. The transfection efficiency of highly fluorinated FP2ks was not severely affected in serum condition, assuming their good serum-compatibility. Discrepancy between their higher cellular uptake efficiency and lower transfection efficiency than PEI25k was thought to arise from the formation of compact polyplexes followed by the decreased dissociation of pDNA. It was also suggested that multiple energy-dependent cellular uptake mechanisms and endosome buffering would mediate the transfection of FP2ks.

Cholic Acid-Conjugated Methylcellulose-Polyethylenimine Nano-Aggregates for Drug Delivery Systems.

Cholic acid-conjugated methylcellulose-polyethylenimines (MCPEI-CAs) were synthesized and characterized for drug delivery systems. Their synthesis was confirmed by 1H NMR and FT-IR analysis. Induced circular dichroism result with Congo red showed that methylcellulose (MC) and polyethylenimine-grafted cationic derivative (MC-PEI) would have helical conformation and random coil structure, respectively. It was found that MCPEI-CAs could form positively charged (>30 mV Zeta-potential) and spherical nano-aggregates (~250 nm Z-average size) by hydrophobic interaction of CA moieties. Critical aggregation concentration of MCPEI-CA10 was measured as 7.2 × 10−3 mg/mL. MCPEI-CA10 could encapsulate the anticancer drug doxorubicin (Dox) with 58.0% of drug loading content and 23.2% of drug loading efficiency and its release was facilitated in acidic condition. Cytotoxicity of MCPEI-CAs was increased with the increase of cholic acid (CA) graft degrees, probably due to the cellular membrane disruption by interaction with specific molecular structure of amphiphilic MCPEI-CA nano-aggregates. MCPEI-CA10/Dox nano-aggregates showed concentration-dependent anticancer activity, which could overcome the multidrug resistance of cancer cells. In this work, molecular conformation change of MC derivatives by chemical modification and a potential of MCPEI-CA10/Dox nano-aggregates for drug delivery systems were revealed.

Structural and functional characterization of the membrane-permeabilizing activity of Nicotiana occidentalis defensin NoD173 and protein engineering to enhance oncolysis.

Defensins are an extensive family of host defense peptides found ubiquitously across plant and animal species. In addition to protecting against infection by pathogenic microorganisms, some defensins are selectively cytotoxic toward tumor cells. As such, defensins have attracted interest as potential antimicrobial and anticancer therapeutics. The mechanism of defensin action against microbes and tumor cells appears to be conserved and involves the targeting and disruption of cellular membranes. This has been best defined for plant defensins, which upon binding specific phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid, form defensin-lipid oligomeric complexes that destabilize membranes, leading to cell lysis. In this study, to further define the anticancer and therapeutic properties of plant defensins, we have characterized a novel plant defensin, Nicotiana occidentalis defensin 173 (NoD173), from N. occidentalis. NoD173 at low micromolar concentrations selectively killed a panel of tumor cell lines over normal primary cells. To improve the anticancer activity of NoD173, we explored increasing cationicity by mutation, with NoD173 with the substitution of Q22 with lysine [NoD173(Q22K)], increasing the antitumor cell activity by 2-fold. NoD173 and the NoD173(Q22K) mutant exhibited only low levels of hemolytic activity, and both maintained activity against tumor cells in serum. The ability of NoD173 to inhibit solid tumor growth in vivo was tested in a mouse B16-F1 model, whereby injection of NoD173 into established subcutaneous tumors significantly inhibited tumor growth. Finally, we showed that NoD173 specifically targets PIP2 and determined by X-ray crystallography that a high-resolution structure of NoD173, which forms a conserved family-defining cysteine-stabilized-αβ motif with a dimeric lipid-binding conformation, configured into an arch-shaped oligomer of 4 dimers. These data provide insights into the mechanism of how defensins target membranes to kill tumor cells and provide proof of concept that defensins are able to inhibit tumor growth in vivo.-Lay, F. T., Ryan, G. F., Caria, S., Phan, T. K., Veneer, P. K., White, J. A., Kvansakul, M., Hulett M. D. Structural and functional characterization of the membrane-permeabilizing activity of Nicotiana occidentalis defensin NoD173 and protein engineering to enhance oncolysis.

The MarR Family Regulator BmrR Is Involved in Bile Tolerance of Bifidobacterium longum BBMN68 via Controlling the Expression of an ABC Transporter.

In order to colonize the human gastrointestinal tract and exert their beneficial effects, bifidobacteria must effectively cope with toxic bile salts in the intestine; however, the molecular mechanism underlying bile tolerance is poorly understood. In this study, heterologous expression of a MarR family transcriptional regulator, BmrR, significantly reduced the ox bile resistance of Lactococcus lactis NZ9000, suggesting that BmrR might play a role in the bile stress response. In silico analysis combined with reverse transcription-PCR assays demonstrated that bmrR was cotranscribed with bmrA and bmrB, which encoded multidrug resistance (MDR) ABC transporters. Promoter prediction and electrophoretic mobility shift assays revealed that BmrR could autoregulate the bmrRAB operon by binding to the bmr box (ATTGTTG-6nt-CAACAAT) in the promoter region. Moreover, heterologous expression of bmrA and bmrB in L. lactis yielded 20.77-fold higher tolerance to 0.10% ox bile, compared to the wild-type strain. In addition, ox bile could disrupt the DNA binding activity of BmrR as a ligand. Taken together, our findings indicate that the bmrRAB operon is autoregulated by the transcriptional regulator BmrR and ox bile serves as an inducer to activate the bile efflux transporter BmrAB in response to bile stress in Bifidobacterium longum BBMN68.IMPORTANCE Bifidobacteria are natural inhabitants of the human intestinal tract. Some bifidobacterial strains are used as probiotics in fermented dairy production because of their health-promoting effects. Following consumption, bifidobacteria colonize the lower intestinal tract, where the concentrations of bile salts remain nearly 0.05% to 2.0%. Bile salts, as detergent-like antimicrobial compounds, can cause cellular membrane disruption, protein misfolding, and DNA damage. Therefore, tolerance to physiological bile stress is indeed essential for bifidobacteria to survive and to exert probiotic effects in the gastrointestinal tract. In B. longum BBMN68, the MarR-type regulator BmrR was involved in the bile stress response by autoregulating the bmrRAB operon, and ox bile as an inducer could increase the expression of the BmrAB transporter to enhance the bile tolerance of BBMN68. Our study represents a functional analysis of the bmrRAB operon in the bile stress response, which will provide new insights into bile tolerance mechanisms in Bifidobacterium and other bacteria.

Bacterial inactivation and in situ monitoring of biofilm development on graphene oxide membrane using optical coherence tomography

In an attempt to advance GO-based environmental applications, herein we probed the anti-biofouling properties and mechanisms of graphene oxide (GO) surface coating. A flexible and mechanically stable GO membrane was fabricated using vacuum filtration technique and its ability to inactivate bacterial growth and subsequent biofilm formation was investigated. Our preliminary results authenticate that the GO membrane, owing to its unique physicochemical surface properties, exhibits superior antibacterial activity against planktonic cell proliferation. An optical coherence tomography (OCT)-based nondestructive in situ monitoring of bacterial biofilm evolution and behavior revealed that the GO surface initially inhibited biofilm growth for 24 h under continuous flow conditions but was incapable of completely averting biofilm development under long-term operation (48 h). We further confirmed that the observed biofilm on the GO membrane was highly unstable and reversibly attached and could be conveniently removed from the surface under a gentle rinsing. Finally, we confirmed that upon direct bacterium-GO contact, GO simultaneously induces cellular membrane disruption and oxidative stress followed by degradation/release of intracellular organelles, thereby causing bacterial inactivation or cell death. We believe our findings will offer new understandings into the anti-biofouling properties of GO-coated membranes and highlight their potential for practical application in membrane-based water and wastewater treatment technologies.

From Antimicrobial Peptides to Antimicrobial Poly(α-amino acid)s.

Conventional small-molecule antibiotics are facing a significant challenge of the rapidly developed drug resistance of pathogens. In contrast, antimicrobial peptides (AMPs), an important component for innate host defenses, are now under intensive investigation as a promising antimicrobial agent for combating drug resistant pathogens. Most AMPs can effectively kill a broad spectrum of pathogens via physical disruption of microbial cellular membranes, which is identified to be difficult to develop resistance. However, the clinical applications of AMPs are still greatly limited by several inherent impediments, such as high cost of production, potential hemolysis or toxicity, and liability to proteinase degradation. Recently, cationic poly(α-amino acid)s with structures mimicking the AMPs are found to have excellent antimicrobial activity. These polymers, termed "antimicrobial poly(α-amino acid)s (APAAs)," have some advantages over AMPs, such as easy production and modification, prolonged antimicrobial activity, low cytotoxicity, and enhanced stability to protease degradation. Here, a brief introduction of mechanisms and affecting factors of microbial killing by AMPs is first presented, followed by a systematic illustration of recent advances in design and preparation of biomimetic APAAs and a perspective in this field.

CAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers.

The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [Keq] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ, resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers.IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

Clinical study to evaluate the performance of a noninvasive focused ultrasound device for thigh fat and circumference reduction compared to control.

An FDA-cleared focused ultrasound device (UltraShape, Syneron Candela® , Yokneam, Israel) for noninvasive abdominal fat reduction produces localized mechanical cellular membrane disruption in adipocytes. This study seeks to determine the safety and efficacy of this device for use on the thighs. Fourteen women aged 33-60 were selected to receive 3 biweekly treatments to one thigh with the other thigh serving as an internal control. The subjects had a BMI range of 18-30kg/m2 and a weight range of 54-83kg. After the third treatment, patients were followed at 4, 8, and 16weeks. Fat thickness was measured by both caliper and ultrasound. In addition, thigh circumference and the patient's weight were measured. Pain, edema, erythema, and adverse events as well as investigator and patient overall satisfaction were recorded at all visits. In comparison with the control, there was a statistically significant average reduction in fat thickness measured by calipers at all time points with a 22.20% (P=.0165) improvement in 16weeks. By ultrasound, there was a 19.23% (4.03mm P=.0051) reduction in fat thickness at 16weeks with statistically significant improvement at the other follow-up visits. At 16weeks, thigh circumference improved, on average, 2.8cm (P=.0059) at the midline. 90.0% of the subjects were satisfied with the results at 16weeks, and the investigator was 100% satisfied. No adverse events were reported; no edema was observed in any subject. All subjects experienced mild erythema. All reported zero pain on a 0-10 scale. Focused ultrasound is safe, effective, and well tolerated to improve the circumference and fat thickness of the thighs without significant side effects. There were no significant adverse events. Investigators and subjects were highly satisfied with the results.

Mechanistic insight into the in vitro toxicity of graphene oxide against biofilm forming bacteria using laser-induced breakdown spectroscopy.

While the cytotoxicity of graphene oxide (GO) has been well established, its bactericidal mechanism, however, has yet to be elucidated to advance GO-based biomedical and environmental applications. In an attempt to better understand the bactericidal action of GO, herein we studied the interactions of GO with Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus cells using physical techniques and chemical probes, respectively. In particular, a novel laser-induced breakdown spectroscopy (LIBS) based elemental fingerprint analysis revealed notable differences between viable and non-viable cells based on the difference in the concentration of trace inorganic elements in complex bacterial systems, which reflect cellular membrane integrity. Lower emission intensities from essential inorganic ions in the GO-treated cells offered explicit evidence on the efflux of intracellular molecules from the bacteria through damaged cell membranes. Furthermore, a detailed structural and morphological investigation of bacterial membrane integrity confirmed GO-induced membrane stress upon direct contact interactions with bacterial cells, resulting in the disruption of cellular membranes. Moreover, the generation of intracellular reactive oxygen species (ROS) in the presence of an added antioxidant underlined the role of GO-mediated oxidative stress in bacterial cell inactivation. Thus, by correlating the changes in the bacterial elemental compositions with the severe morphological alterations and the high ROS production witnessed herein, we propose that the bactericidal mechanism of GO is likely to be the synergy between membrane and oxidative stress towards both tested species. Our findings offer useful guidelines for the future development of GO-based antibacterial surfaces and coatings.

Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique.

Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP and the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples.

Membrane Oxidation in Cell Delivery and Cell Killing Applications.

Cell delivery or cell killing processes often involve the crossing or disruption of cellular membranes. We review how, by modifying the composition and properties of membranes, membrane oxidation can be exploited to enhance the delivery of macromolecular cargoes into live human cells. We also describe how membrane oxidation can be utilized to achieve efficient killing of bacteria by antimicrobial peptides. Finally, we present recent evidence highlighting how membrane oxidation is intimately engaged in natural biological processes such as antigen delivery in dendritic cells and in the killing of bacteria by antimicrobial peptides. Overall, the insights that have been recently gained in this area should facilitate the development of more effective delivery technologies and antimicrobial therapeutic approaches.

RIP1 and RIP3 contribute to shikonin-induced DNA double-strand breaks in glioma cells via increase of intracellular reactive oxygen species

Shikonin has been reported to induce glioma cell death via necroptosis, a type of programmed necrosis primarily mediated by RIP1 and RIP3. Although RIP1 and RIP3 are found to regulate some features of necrosis such as energy depletion and cellular membrane disruption, it remains unclear whether RIP1 and RIP3 could modulate DNA double strand breaks (DSBs), which is a crucial event leading to chromatinolysis. In this study, we used glioma cell lines and mice model of xenograft glioma to investigate the roles of RIP1 and RIP3 in shikonin-induced DNA DSBs. We found that shikonin induced upregulation of RIP1 and RIP3, necrosome formation and DNA DSBs in vitro and in vivo. In vitro investigation showed that the DNA DSBs and the reduction of cellular viabilities induced by shikonin were both prevented when RIP1 or RIP3 was pharmacologically inhibited by specific inhibitor or genetically knocked down with siRNA. Then, we proved that suppression of intracellular ROS with antioxidant NAC inhibited DNA DSBs caused by either hydrogen peroxide or shikonin, suggesting that ROS played a crucial role in regulation of DNA DSBs of glioma cells induced by shikonin. Further, we found that RIP1 and RIP3 regulated shikonin-induced overproduction of ROS via causing excessive generation of mitochondrial superoxide and depletion of GSH, indicating that ROS was the downstream signal of RIP1 and RIP3. Taken together, we demonstrated that RIP1 and RIP3 contributed to shikonin-induced DNA DSBs in glioma cells via increase of intracellular ROS levels.

Preclinical Evidence on the Anticancer Properties of Food Peptides.

Natural, synthetic and analogues of peptides have shown prospects for application in cancer chemotherapy. Notably, some food protein-derived peptides are known to possess anticancer activities in cultured cancer cells, and also in animal cancer models via different mechanisms including induction of apoptosis, cell cycle arrest, cellular membrane disruption, inhibition of intracellular signalling, topoisomerases and proteases, and antiangiogenic activity. Although the mechanism of several anticancer food peptides is yet to be clearly elucidated, there is potential for practical applications of the peptides as functional food and nutraceutical ingredients, especially in adjuvant cancer therapy. This review describes the aetiological mechanisms of cancers and the production, structures, mechanisms of action, availability, and cellular and physiological anticancer activities of the food peptides.

Cell membrane disruption stimulates cAMP and Ca2+ signaling to potentiate cell membrane resealing in neighboring cells.

ABSTRACTDisruption of cellular plasma membranes is a common event in many animal tissues, and the membranes are usually rapidly resealed. Moreover, repeated membrane disruptions within a single cell reseal faster than the initial wound in a protein kinase A (PKA)- and protein kinase C (PKC)-dependent manner. In addition to wounded cells, recent studies have demonstrated that wounding of Madin-Darby canine kidney (MDCK) cells potentiates membrane resealing in neighboring cells in the short-term by purinergic signaling, and in the long-term by nitric oxide/protein kinase G signaling. In the present study, real-time imaging showed that cell membrane disruption stimulated cAMP synthesis and Ca2+ mobilization from intracellular stores by purinergic signaling in neighboring MDCK cells. Furthermore, inhibition of PKA and PKC suppressed the ATP-mediated short-term potentiation of membrane resealing in neighboring cells. These results suggest that cell membrane disruption stimulates PKA and PKC via purinergic signaling to potentiate cell membrane resealing in neighboring MDCK cells.

Cryopreservation and xenografting of human ovarian fragments: medulla decreases the phosphatidylserine translocation rate.

BackgroundPhosphatidylserine is the phospholipid component which plays a key role in cell cycle signaling, specifically in regards to necrosis and apoptosis. When a cell affected by some negative factors, phosphatidylserine is no longer restricted to the intracellular side of membrane and can be translocated to the extracellular surface of the cell. Cryopreservation can induce translocation of phosphatidylserine in response to hypoxia, increasing intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species and lipid peroxidation. As such the aim of this study was to test the level of phosphatidylserine translocation in frozen human medulla-contained and medulla-free ovarian tissue fragments.MethodsOvarian fragments from twelve patients were divided into small pieces of two types, medulla-free cortex (Group 1, n = 42, 1.5–3.0 × 1.5–3.0 × 0.5–0.8 mm) and cortex with medulla (Group 2, n = 42, 1.5–3.0 × 1.5–3.0 × 1.5–2.0 mm), pre-cooled after operative removal to 5 °C for 24 h and then conventionally frozen with 6 % dimethyl sulfoxide, 6 % ethylene glycol and 0.15 M sucrose in standard 5-ml cryo-vials. After thawing at +100 °C and step-wise removal of cryoprotectants in 0.5 M sucrose, ovarian pieces were xenografted to SCID mice for 45 days. The efficacy of tissues cryopreservation, taking into account the presence or absence of medulla, was evaluated by the development of follicles (histology with hematoxylin-eosin) and through the intensity of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide).ResultsFor Groups 1 and 2, the mean densities of follicles per 1 mm3 were 9.8, and 9.0, respectively. In these groups, 90 and 90 % preantral follicles appeared morphologically normal. However, FACS analysis showed a significantly decreased intensity of translocation of phosphatidylserine (FITC-Annexin V positive) after cryopreservation of tissue with medulla (Group 2, 59.6 %), in contrast with tissue frozen without medulla (Group 1, 78.0 %, P < 0.05). In Groups 1 and 2 it was detected that 21.6 and 40.0 % cells were viable (FITC-Annexin V negative, Propidium Iodide negative).ConclusionThe presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue.