A comparative study of the effectiveness of cisplatin and 5-fluorouracil on cutaneous squamous human carcinoma cell line: Potential chemotherapy alternative to surgery.

Surgery as treatment for local invasive cutaneous squamous cell carcinoma (cSCC) is not always feasible due to the age and/or the health status of patients. Thus, the investigation of new strategies to improve the quality of life of them is required. The aim of this work is to investigate two chemotherapy agents individually on cSCC cells with the purpose to provide a better understanding of the effectiveness underlying each one. The cisplatin effectiveness is compared at different times with that observed for the 5-fluorouracil treatment. The effectiveness of both was assessed by using flow cytometry to determine the survival cell ratio, and QBlue test to study the cell recovery ability after treatments. A significant increase in the number of apoptotic cells, especially 48 hours after treatments, has been detected. Despite this, cisplatin arises as the most promising agent for the treatment of local invasive cutaneous squamous cell carcinoma due to the fact that a lower concentration and time are required to observe a higher effectiveness on cells with respect to the 5-fluorouracil. An optimal cisplatin-based chemotherapy might provide a better outcome for patients affected by a local invasive cSCC rather than surgery.

Antioxidant activity of N-acetylcysteine, flavonoids and alpha-tocopherol on endometrial cells in culture.

An appropriate local environment is necessary for successful implantation. Oxidative stress is implicated in the pathogenesis of several pathologies, and may contribute to early pregnancy failure. Antioxidant therapies have been studied in infertility. In this study, we have assessed the antioxidant activity of N-acetylcysteine (NAC), flavonoids (quercetin, catechin) and alpha-tocopherol in an oxidative model of endometrial cells (RL95). Endometrial cells were incubated at several hydrogen peroxide concentrations. Antioxidant effects of NAC (15 mM), quercetin (150 microM), catechin (150 microM) and alpha-tocopherol included in liposomes (1.6 microg) were assessed by measuring cell viability by the MTT assay. Alpha-tocopherol-liposomes taken up by endometrial cells were assessed by HPLC. All liposomes used were able to introduce alpha-tocopherol into cells. The antioxidant effect of NAC and quercetin improved the viability of oxidised cells, and this effect was observed when the oxidant and antioxidant were coincubated. No viability change occurred when the antioxidant was added before or after the oxidant. The antioxidant effect of NAC was better than that of quercetin. When catechin or alpha-tocopherol were used in the same conditions, no antioxidant effect was detected in cells in culture. These results demonstrate that NAC and quercetin are good H2O2 scavengers.

Induction of the blue form of bacteriorhodopsin by low concentrations of sodium dodecyl sulfate.

The effects produced on bacteriorhodopsin by low concentrations of several detergents have been studied by absorption and fourth-derivative spectrophotometry. Sodium dodecyl sulfate induces the appearance of the blue form of bacteriorhodopsin (lambda max = 600 nm) at pH values up to 7.0 in a reversible manner. The apparent pK of the purple-to-blue transition raised with increasing concentration of SDS. Of the other detergents tested, only sodium dodecyl-N-sarcosinate showed a slight red-shift of the absorption band to 580 nm, whereas sodium taurocholate, Triton X-100 and cetyltrimethylammonium bromide did not favour the appearance of the blue form. The effect of SDS was found to be consistent with a localized conformational change that moves away the counter-ion of the protonated Schiff base.

Thr90 is a key residue of the bacteriorhodopsin proton pumping mechanism.

Mutation of Thr90 to Ala has a profound effect on bacteriorhodopsin properties. T90A shows about 20% of the proton pumping efficiency of wild type, once reconstituted into liposomes. Mutation of Thr90 influences greatly the Schiff base/Asp85 environment, as demonstrated by altered lambda(max) of 555 nm and pK(a) of Asp85 (about 1.3 pH units higher than wild type). Hydroxylamine accessibility is increased in both dark and light and differential scanning calorimetry and visible spectrophotometry show decreased thermal stability. These results suggest that Thr90 has an important structural role in both the unphotolysed bacteriorhodopsin and in the proton pumping mechanism.

Biopolymer excreted by pseudoalteromonas antarctica NF(3), as a coating and protective agent of liposomes against dodecyl maltoside

The ability of an exopolymer of glycoproteic character (GP) excreted by a new gram-negative species Pseudoalteromonas antarctica NF(3), to coat phosphatidylcholine (PC) liposomes and to protect these bilayers against the action of the nonionic surfactant dodecyl maltoside was investigated. Transmission electron microscopy (TEM) micrographs of freeze fractured liposome/GP aggregates reveal that the addition of the glycoprotein to liposomes led to the formation of a film (polymer adsorbed onto the bilayers) that tightly coated PC bilayers. The complete coating was already achieved at a PC : GP weight ratio of about 9:1. Image analysis profiles of digitalized TEM micrographs (PC : GP weight ratio 8:2) show that this film was formed by a multilayer structure. The periods of the average distance of the pattern ordering in layer structures (9-10 layers) were of about 2-3 nm and the thickness of the complete film was of about 25 nm. Higher amounts of glycoprotein resulted in a growth of this film, which exhibited at the highest proportion of this compound (50% in weight) a multifilm structure. An increasing resistance of liposomes to be affected by dodecyl maltoside both at subsolubilizing and solubilizing levels occurred as the proportion of the glycoprotein in the system rose, although this protective effect was more effective at low proportions of this compound (PC : GP weight ratios from 9:1 to 8:2). Thus, although a direct dependence was found between the growth of the enveloping structure and the resistance of the coated liposomes to be affected by the surfactant, the more effective protection occurred when this structure was a thin film formed by the assembly of various layers of GP of about 2-3 nm. Copyright 1999 John Wiley & Sons, Inc.

Conformational isomerizations of the poly(dA-dT) and poly(amino2dA-dT) duplexes involving the unusual X-DNA double helix: a fourth derivative spectrophotometric study.

Fourth derivative spectrophotometry has been applied to monitor conformational isomerizations of polynucleotides for the first time. The transitions studied have been the B-A and A-X isomerizations of poly(dA-dT) and the B-X one of poly(amino2dA-dT). Parameters obtained from the fourth derivative spectra have been used to follow these conformational changes. The A form of poly(dA-dT) has been characterized by a new fourth derivative peak at 293.0 nm which can be associated to interstrand adenine-adenine interactions. Furthermore, some of the fourth derivative peaks in the long wavelength region (270-310 nm) can be related to stacking interactions present in the polynucleotide double helices. The tentative assignment of these peaks, particularly that at 299.0 nm in the derivative spectra of poly(amino2dA-dT), to n----pi electronic transitions is discussed.

2-Hydroxy-5-nitrobenzyl bromide as a specific reagent for tryptophan residues in membrane proteins: bacteriorhodopsin as an example.

The use of 2-hydroxy-5-nitrobenzyl bromide for the modification of tryptophan residues in integral membrane proteins is exemplified by its application to bacteriorhodopsin from Halobacterium halobium. Complete elimination of the unreacted reagent requires delipidation of the sample with detergents and posterior chromatography. This method also allows separation of the modified from the unmodified bacteriorhodopsin molecules. Modified molecules have lost the retinal, and are thus bleached, whereas the unmodified molecules appear to retain all the characteristics of solubilized native bacteriorhodopsin.

Factors involved in the production of liposomes with a high-pressure homogenizer.

A variety of factors were evaluated in order to establish the characteristics of the liposomes obtained with a high-pressure homogenizer (Microfluidizer 110S). The experimental mean diameter of the liposomes and the width of their size distribution were correlated to surfaces calculated from the responses obtained by the combination of two groups of factors. First, the inlet pressure of the homogenizer, the times that the samples were processed (cycles) and the bulk ionic strength. Second, the phospholipid (P) and ethanol concentrations. The variation of the entrapped liposome volume upon the pressure and cycles was also studied. All the calculated surfaces are statistically significant and have a low S.E. of estimate. Mean liposome diameter decreases with increasing inlet pressure, number of cycles and ethanol concentration, and increases raising ionic strength. No P concentration effect was observed. The variation of the entrapped volume of liposomes upon the cycles and pressure has a similar behavior to the diameter. Within the studied variable range, microfluidization does not increase the P oxidation index, nor does it significantly alter the P concentration.

Use of gamma-inulin/liposomes/Vitamin E adjuvant combination in contraceptive vaccines.

The adjuvanticity of two gamma inulin/liposomes/Vitamin E combinations was evaluated in the mouse, in contraceptive vaccines with sperm protein extracts or a synthetic HE2 peptide ("Human Epididymis gene product"; residues 15-28) as antigen. The HE2 peptide was not conjugated to a protein carrier, to ensure that the antibodies elicited were specific against the HE2 peptide. The adjuvant combinations were designed to increase adjuvanticity, as their components have complementary mechanisms, and their performance was compared to Freund's adjuvant. Antibody production against native sperm structures was determined in sera by ELISA immunoassay and immunohistology. Toxicity of adjuvants was determined by histopathological study and treated mice were monitored for signs of pain or distress. Our results show that the gamma inulin (1-2 microm particle size)/liposomes/Vitamin E combination, with sperm protein extracts, is better than Freund's adjuvant because it elicits good antibody titres without any toxicity. When the synthetic HE2 peptide is used as antigen, the gamma inulin (1-2 microm particle size)/liposomes/Vitamin E combination is less effective than Freund's adjuvant; nevertheless, the anti-HE2 antibodies elicited are highly specific and recognize native structures in sperm.

Double-strand breaks on F98 glioma rat cells induced by minibeam and broad-beam synchrotron radiation therapy.

PURPOSE: To assess the DNA damage induced by MBRT and BB radiations on glioma cells. METHODS: The analysis of fluorescent intensity emitted per nucleus was plotted versus DNA content 2 and 17 h after irradiations. At around cell-doubling time (17 h) after exposures, the remaining DNA radiation damage could be correlated with cellular death. RESULTS: A higher γH2AX IF intensity per cell could be detected 2 and 17 h after MBRT when compared with BB. 17 h after MBRT, misrepaired damaged cells remained arrested in both G1 and G2 phases. CONCLUSIONS: A pronounced G2 phase arrest was detected at 17 h after MBRT and BB. However, only after MBRT, a dose-dependent increasing number of damaged cells appeared arrested also in the G1 phase, and a higher amount of cells more prone to undergo apoptosis were detected. The threshold dose required to enhance the effectiveness of both synchrotron radiation techniques was 12 Gy.

Synchrotron radiation in cancer treatments and diagnostics: an overview.

During the last 30 years many groups have carried out experiments and trials to develop new imaging and radiotherapy techniques in oncology, based on the use of synchrotron X-rays. There are several synchrotron biomedical stations around the world, which offer an excellent platform to improve either the imaging diagnosis or radiotherapy treatment for different tumour types. In the coming months the first radiotherapy clinical trials will be seen at the Biomedical Beamline at the ESRF synchrotron in Grenoble (France). In this article we highlight the results of some of the techniques and strategies that have been developed at different biomedical synchrotron stations.

Fourier transform infrared analysis of bacteriorhodopsin secondary structure.

Fourier transform infrared spectroscopy at a resolution of 1 cm-1 has been used to study the conformation of dark-adapted bacteriorhodopsin in the native purple membrane, in H2O and D2O suspensions. A detailed analysis of the amide I bands was made using derivative and deconvolution techniques. Curve-fitting results of four independent experiments indicate, after estimation of the methodological errors, that native bacteriorhodopsin contains 52-73% alpha-helices, 13-19% reverse turns, 11-16% beta-sheets, and 3-7% unordered segments. Our analysis has enabled the identification of several components corresponding to alpha-helices, beta-sheets, and reverse turns. Besides the alpha I- and alpha II-helices (peaking at 1658 and 1665 cm-1), we propose that two more infrared bands arise from alpha-helical structures: one at 1650 cm-1 from alpha I and another one at 1642 cm-1 in H2O suspension, which could originate from type III beta-turns (i.e., one turn of 3(10)-helix). The relatively high content of reverse turns suggests the presence of one reverse turn per loop, plus another one in the C-terminal segment. On the other hand, several reasons argue that the calculated mean beta-sheet content of around 14% should be decreased somewhat. These beta-sheets could be located in the noncytoplasmatic links of the bacteriorhodopsin molecule.

Fourth-derivative spectrophotometry analysis of tryptophan environment in proteins. Application to melittin, cytochrome c and bacteriorhodopsin.

Fourth-derivative spectrophotometry is applied to the analysis of solvent effects on the spectral transitions of N-acetyl-L-tryptophan amide, as well as of its mixtures with N-acetyl-L-tyrosine ethyl ester. These compounds were analyzed in different media. It was found that the position of the longest-wavelength minimum of the fourth-derivative spectrum is mainly determined by the nature of the Trp environment, with a minor contribution from that of Tyr. A geometrical parameter is also defined, which depends on both Trp and Tyr environments. The simultaneous consideration of both parameters allows an estimation to be made of the Trp environment. The method is applied to the study of conformational changes of three well-characterized proteins: melittin from bee venom, cytochrome c from horse heart and bacteriorhodopsin from Halobacterium halobium. The results obtained are found to be in accordance with the known conformational properties of these proteins. In addition, the fourth-derivative operation completely eliminates the interference from the near-ultraviolet bands of the prosthetic groups.

The role of retinal in the thermal stability of the purple membrane.

Differential scanning calorimetry demonstrates that the bleached form of the purple membrane does not possess any measurable thermal transition in water, up to 105 degrees C, whereas in 0.1 M phosphate pH 7.5 it shows a transition at about 82 degrees C, with an enthalpy of 110 kJ/mol. In the latter medium, the native membrane shows the main transition at 97 degrees C, with an enthalpy of 390 kJ/mol. The reduced form of the purple membrane shows two small transitions in water, as well as in 0.1 M phosphate, which do not seem to be related to the main thermal transition of the native membrane. Fourier-transform infrared spectra in D2O show that the two modified samples, as well as the native one, undergo similar secondary structural changes upon thermal denaturation. These changes appear to extend through a wide temperature range for both modified forms, particularly for the bleached one. The results suggest that the main thermal transition in the purple membrane is due to a cooperative conformational change involving the disruption of the network of electrostatic and hydrogen-bonding interactions which originate from the protonated Schiff base. In the two modified membranes, these conformational changes appear to proceed smoothly through a rather low or non-cooperative process. The thermal behaviour of the bleached membrane in water resembles that of the molten globule state described for several globular proteins.

Assembly properties and applications of a new exopolymeric compound excreted by Pseudoalteromonas antarctica NF3.

The self assembly properties and applications of an exopolymeric compound (EC) of a glycoprotein character excreted by a new gram-negative species, Pseudoalteromonas antarctica NF3, have been reviewed. This compound exhibited surface-active properties in water, with a concentration of 0.20 mg ml(-1) being the key value associated with its physicochemical properties. Unsonicated EC aqueous dispersions showed the coexistence of concentric multilamellar and small unilamellar aggregates by transmission electron microscopy (TEM). Sonication of these dispersions revealed that each lamellae of the initial multilamellar structures were made up of various subunits coiled coils. As for the ability of this exopolymeric biomaterial to coat phosphatidylcholine (PC) liposomes and to protect these vesicles against different surfactants, freeze-fracture TEM micrographs of liposome/EC aggregates revealed that the addition of the EC to liposomes led to the formation of a film (polymer adsorbed onto the bilayers) that coated very well the PC bilayers. The complete coating was already achieved at a PC:EC weight ratio of about 9:1. An increasing resistance of PC liposomes to surfactants (in particular sodium dodecyl sulfate) occurred as the proportion of EC in the system rose, although this effect was more effective at low EC proportions (PC:EC weight ratios from 9:1 to 8:2). Although a direct dependence was found between the growth of the enveloping structure and the resistance of the coated liposomes to be affected by the surfactants, the best protection occurred when this structure was a thin film of about 20-25 nm formed by nine to ten layers of about 2-3 nm.

Ability of the exopolymer excreted by Pseudoalteromonas antarctica NF3, to coat liposomes and to protect these structures against octyl glucoside.

The ability of an exopolymer of glycoproteic character (GP) excreted by a new gram-negative specie Pseudoalteromonas antarctica NF3, to coat phosphatidylcholine (PC) liposomes and to protect these bilayers against the action of the nonionic surfactant octyl glucoside (OG) has been investigated. TEM micrographs of freeze-fractured liposome/GP aggregates reveal that the addition of GP to liposomes led to the formation of a covering structure (polymer adsorbed onto the bilayers) that tightly coated PC bilayers. The complete coating was already achieved when the proportion of GP assembled with liposomes was approximately 10% (wt% vs total PC). Higher GP amounts resulted in a growth of this coating structure which exhibited at the highest GP proportion in the system (31% of assembled GP) a multilayered structure. An increasing resistance of PC liposomes to be affected by OG both at sublytic and lytic levels occurred as the proportion of GP in the system rose; this protective effect being more effective when the proportion of assembled GP was 10-20% in weight. Thus, although a direct dependence was found between the growth of the enveloping structure and the resistance of the coated liposomes to be affected by OG, the best protection occurred when the proportion of assembled GP was about 10 wt%.