Candida albicans inactivation and cell membrane integrity damage by microwave irradiation

In indicating the microwave irradiation for disinfecting dentures it is necessary to see how this procedure influences Candida albicans integrity and viability. The aim of this study was to evaluate the ability of microwaves to inactivate C. albicans and damage cell membrane integrity. Two 200-ml C. albicans (ATCC 10231) suspensions were obtained. A sterile denture was placed in a beaker containing the Experimental (ES) or the Control suspension (CS). ES was microwaved at 650 W for 6 min. Suspensions were optically counted using methylene blue dye uptake as indicative of membrane-damaged cells; spread on Agar Sabouraud dextrose (ASD) for viability assay; or spectrophotometrically measured at 550 nm. Cell-free solutions were submitted to content analyses of protein (Bradford and Pyrogallol red methods); Ca++ (Cresolftaleine complexone method); DNA (spectrophotometer measurements at 260 nm) and K + (selective electrode technique). Data were analysed by Student's t- or Wilcoxon z-tests (α = 0.05). All ES cells demonstrated cell membrane damage. Viable cells were non-existent in the ES ASD plates. No significant difference in optical density between ES and CS was observed (P = 0.272). ES cells released significantly high protein (P < 0.001, Bradford; P = 0.005, Pyrogallol red), K+ (P < 0.001), Ca++ (P = 0.012) and DNA (P = 0.046) contents. Microwaves inactivated C. albicans and damaged cell membrane integrity. © 2007 The Authors.

Cell membrane integrity of candida albicans after different protocols of microwave irradiation

To evaluate the ability of low time microwaveexposureto inactivate and damage cell membrane integrity of C. albicans. Materials and Methods: Two 200ml C. albicans suspensions were obtained. Sterile dentures were placed in a beaker containing Experimental (ES) or Control suspensions (CS). ES was microwaved at 650 W for 1, 2, 3, 4 or 5 min. Suspensions were optically counted using Methylene blue dye as indicative of membrane-damaged cells; spread on Agar Sabouraud dextrose (ASD) for viability assay; or spectrophotometrically measured at 550nm. Cell-free solutions were submitted to content analyses of protein (Bradford and Pyrogallol red methods); Ca++ (Cresolphthalein Complexone method); DNA (spectrophotometer measurements at 260nm) and K+ (selective electrode technique). Data were analyzed by Student-t test and linear regression (α=0.05). In addition, flowcytometry analysis of Candida cells in suspensionwas performed using propidium iodide. Results: All ES cells demonstrated cell membrane damage at 3, 4 and 5 min,viable cells were nonexistent at 3, 4 and 5 min ES ASD plates and optical density of ES and CS was not significantly differentfor all exposition times. ES cells released highcontents of protein, K+ , Ca++ and DNA after 2 min exposition when compared to that of the CSs. Similar results were observed with flow cytometry analysiswith regard to the periodsof microwave exposure. Conclusions: Microwave irradiation inactivated C. albicansafter 3min and damaged cell membrane integrity after 2 min exposition.

Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3h to (14)C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 microM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (=low accumulation compounds). DBP and DDE showed much higher cellular accumulations (=high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.

Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: study of angiogenic signaling by ephrin-B2

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.

Transbilayer Phospholipid Asymmetry in Plasmodium knowlesi- Infected Host Cell Membrane

The membranes from normal and Plasmodium knowlesi-infected rhemsus monkey erythrocytes (90 to 95 percent infected with early ring stage) were analyzed for transbilayer distribution of phosphatidylcholine (PC). hosphatidylethanolamine (PE). and hosphatidylserine (PS). by means of chemical and enzymatic probes. The external monolayer of the normal red cell membrane contained at least 68 to 72 percent of the total phosphatidylcholine and 15 to 20 percent of the total phosphati dylethanolamine. In the infected cell, the transmembrane phosphatidylcholine distribution appeared to be reversed, with only 20 to 30 percent of it being externally localized, whereas roughly equal amounts of phosphatidylethanolamine were present in the outer and'inner surfaces. However, total pho.~phatid)'lserine in both the infected and normal red cells was exc/usi~'ely internal. Unlike that in the normal intact cell, external phosphatidylethanolamine in the parasitized cell was readily accessible to phospholipase A2. These results indicate that significant changes in molecular architecture of the host cell membrane are the result of varasitization.

THE MECHANISM OF TUMORIGENESIS IN THE IMMORTALIZED HUMAN PANCREATIC CELL LINES: CELL CULTURE MODELS OF HUMAN PANCREATIC CANCER

The mechanism of tumorigenesis in the immortalized human pancreatic cell lines: cell culture models of human pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer in the world. The most common genetic lesions identified in PDAC include activation of K-ras (90%) and Her2 (70%), loss of p16 (95%) and p14 (40%), inactivation p53 (50-75%) and Smad4 (55%). However, the role of these signature gene alterations in PDAC is still not well understood, especially, how these genetic lesions individually or in combination contribute mechanistically to human pancreatic oncogenesis is still elusive. Moreover, a cell culture transformation model with sequential accumulation of signature genetic alterations in human pancreatic ductal cells that resembles the multiple-step human pancreatic carcinogenesis is still not established. In the present study, through the stepwise introduction of the signature genetic alterations in PDAC into the HPV16-E6E7 immortalized human pancreatic duct epithelial (HPDE) cell line and the hTERT immortalized human pancreatic ductal HPNE cell line, we developed the novel experimental cell culture transformation models with the most frequent gene alterations in PDAC and further dissected the molecular mechanism of transformation. We demonstrated that the combination of activation of K-ras and Her2, inactivation of p16/p14 and Smad4, or K-ras mutation plus p16 inactivation, was sufficient for the tumorigenic transformation of HPDE or HPNE cells respectively. We found that these transformed cells exhibited enhanced cell proliferation, anchorage-independent growth in soft agar, and grew tumors with PDAC histopathological features in orthotopic mouse model. Molecular analysis showed that the activation of K-ras and Her2 downstream effector pathways –MAPK, RalA, FAK, together with upregulation of cyclins and c-myc were involved in the malignant transformation. We discovered that MDM2, BMP7 and Bmi-1 were overexpressed in the tumorigenic HPDE cells, and that Smad4 played important roles in regulation of BMP7 and Bmi-1 gene expression and the tumorigenic transformation of HPDE cells. IPA signaling pathway analysis of microarray data revealed that abnormal signaling pathways are involved in transformation. This study is the first complete transformation model of human pancreatic ductal cells with the most common gene alterations in PDAC. Altogether, these novel transformation models more closely recapitulate the human pancreatic carcinogenesis from the cell origin, gene lesion, and activation of specific signaling pathway and histopathological features.

Contraction speed of the actomyosin cytoskeleton in the absence of the cell membrane

The contraction of the actomyosin cytoskeleton, which is produced by the sliding of myosin II along actin filaments, drives important cellular activities such as cytokinesis and cell migration. To explain the contraction velocities observed in such physiological processes, we have studied the contraction of intact cytoskeletons of Dictyostelium discoideum cells after removing the plasma membrane using Triton X-100. The technique developed in this work allows for the quantitative measurement of contraction rates of individual cytoskeletons. The relationship of the contraction rates with forces was analyzed using three different myosins with different in vitro sliding velocities. The cytoskeletons containing these myosins were always contractile and the contraction rate was correlated with the sliding velocity of the myosins. However, the values of the contraction rate were two to three orders of magnitude slower than expected from the in vitro sliding velocities of the myosins, presumably due to internal and external resistive forces. The contraction process also depended on actin cross-linking proteins. The lack of α-actinin increased the contraction rate 2-fold and reduced the capacity of the cytoskeleton to retain internal materials, while the lack of filamin resulted in the ATP-dependent disruption of the cytoskeleton. Interestingly, the myosin-dependent contraction rate of intact contractile rings is also reportedly much slower than the in vitro sliding velocity of myosin, and is similar to the contraction rates of cytoskeletons (different by only 2–3 fold), suggesting that the contraction of intact cells and cytoskeletons is limited by common mechanisms.

Cell membrane fluctuations are regulated by medium macroviscosity: Evidence for a metabolic driving force

Extracellular fluid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body fluids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical fluctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase fluid viscosity. The parameters of cell membrane fluctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane fluctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATP-depleted red blood cells elevation of EMF did not affect cell membrane fluctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane fluctuations are driven by a metabolic driving force in addition to the thermal one.

The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO2 permeability of the red blood cell membrane

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of 12C18O16O from a red cell suspension as the 18O exchanges between labeled CO2 + HCO3− and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO3− (Pm,HCO−3). To test this technique, we added sufficient 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) to inhibit all the HCO3−/Cl− transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced Pm,HCO−3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in Pm,CO2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO2 and HCO3−, we found that DIDS inhibited both Pm,HCO−3 and Pm,CO2, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO2 exchange.