Concentration and distribution of silica nanoparticles in colon cancer cells assessed by synchrotron based X-ray techniques.

The quantitative uptake of Silica nanoparticles (SiNPs), although representing an essential prerequisite for their theranostic use, is difficult to address and it is still not utterly investigated. In this study, we tested the uptake and toxicity of two different types of luminescent core-shell silica-PEG (polyethylene glycol) nanoparticles SiNP and their carboxylate analogues on human adenocarcinoma cell line LoVo. We assessed the intracellular spatial distribution and concentration of Si element in the cell by a state-of-the-art approach merging synchrotron-based X-ray techniques (XRFM) with scanning transmission X-Ray microscopy (STXM). The concentration maps of Si obtained reflect the distribution of the SiNPs. In addition, we calculated the number of SiNPs per volume unit in each single cell, quantitating the exact amount of conveyed particles. The absence of effects on proliferation and cell death was confirmed by viability assays, morphological analysis and cytofluorimetric evaluation of ROS content. The three-dimensional analysis of intracellular uptake of both types of nanoparticles (with different surface charge) was performed by confocal fluorescence microscopy, which showed a main localization in the cytosolic region with no sign of nuclear uptake.

p53-dependent and p53-independent anticancer activity of a new indole derivative in human osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant tumor of bone, occurring most frequently in children and adolescents. The mechanism of formation and development of OS have been studied for a long time. Tumor suppressor pathway governed by p53 gene are known to be involved in the pathogenesis of osteosarcoma. Moreover, loss of wild-type p53 activity is thought to be a major predictor of failure to respond to chemotherapy in various human cancers. In previous studies, we described the activity of a new indole derivative, NSC743420, belonging to the tubulin inhibitors family, capable to induce apoptosis and arrest of the cell cycle in the G2/M phase of various cancer cell lines. However, this molecule has never been tested on OS cell line. Here we address the activity of NSC743420 by examine whether differences in the p53 status could influence its effects on cell proliferation and death of OS cells. In particular, we compared the effect of the tested molecule on p53-wild type and p53-silenced U2OS cells, and on SaOS2 cell line, which is null for p53. Our results demonstrated that NSC743420 reduces OS cell proliferation by p53-dependent and p53-independent mechanisms. In particular, the molecule induces proliferative arrest that culminate to apoptosis in SaOS2 p53-null cells, while it brings a cytostatic and differentiating effect in U2OS cells, characterized by the cell cycle arrest in G0/G1 phase and increased alkaline phosphatase activity.

Seroprevalencia de la enfermedad de Chagas en El Espìnillo y Villa Río Bermejito en la provincia de Chaco / Seroprevalence of Chagas disease in El Espinillo and Villa Río Bermejito in the province of Chaco

En el marco del proyecto "Pediatría ambulatoria itinerante para comunidades rurales y aisladas" (P.A.I.C.R.A.) diagramado por LA HIGUERA.ONG en cooperación con el Laboratorio de Villa Río Bermejito y Sistema de Salud de provincia de Chaco; entre los meses de julio a diciembre de 2007 se realizó el relevamiento serológico en el área programática correspondientes a Villa Río Bermejito y El Espinillo del Monte Impenetrable (Zona Sanitaria VI) de la provincia de Chaco. En este área viven alrededor 9200 personas pertenecientes a comunidades de pueblos originarios (tobas) y criollos, que ocupan un área geográfica de 4000 km2, en la cual las condiciones sociosanitarias son propicias para el desarrollo de esta enfermedad. Se realizaron 1296 serologías para la enfermedad de Chagas con sangre extraída por venopunción. Se utilizaron 3 técnicas de procesamiento diferentes: hemaglutinación indirecta (HAI, Chagas Polycraro S.A.I.C), enzimainmunoensayo (ELISA Chagas Test, Wienner lab) e Inmuno fluorescencia Indirecta (IFI). Se consideraron positivas las muestras que fueron reactivas por dos técnicas serológicas. Se obtuvo evidencia serológica de infección por Tripanosoma Cruzi en 529(40,8%) de 1296 personas, en el grupo de edad de 0-15 años en 191 (29%); y en los menores de 5 años de 44(22,2%). Los datos obtenidos muestran un escaso control vectorial con valores muy similares a estudios previos que se hicieron en esta misma área hace siete años. Un dato preocupante es que el 66,6% de las mujeres en edad fértil está infectada, lo que lleva a predecir un alto riesgo de chagas congénito. Este trabajo demuestra que son imprescindibles políticas sanitarias sostenidas en el tiempo, con equipos de trabajo con funciones preestablecidas destinadas por un lado al control del vector y por otro al diagnóstico, tratamiento y seguimiento en toda el área endémica, por profesionales especializados, preferentemente del área pediátrica (AU)

Within the framework of the project "Itinerary outpatient pediatrics for rural and isolated communities" (P.A.I.C.R.A.) created by LA HIGUERA.ONG in cooperation with the Laboratory of Villa Río Bermejito and the Chaco Health System, a serological survey was conducted in the study area of Villa Río Bermejito and El Espinillo del Monte Impenetrable (Sanitary zone VI) in the province of Chaco between July and December 2007. The area has around 9200 inhabitants belonging to indigenous (tobas) and immigrant-descendant communities and covers 4000 km2 where socio-sanitary conditions favor the spread of Chagas disease. By venipuncture, 1296 blood samples were drawn to be evaluated for Chagas disease. Three different techniques were used: indirect hemagglutination assay (IHA, Chagas Polycraro S.A.I.C), enzyme-linked immunosorbent assay (ELISA Chagas Test, Wienner lab), and indirect immunofluorescence assay (IFA). Samples that showed reactivity in at least two techniques were considered positive. Serological evidence of infection by Trypanosoma cruzi was found in 529 (40.8%) of 1296 persons; 191 (29%) in the age group of 0-15 years and 44 (22.2%) under 5 years of age. The data show poor vectorial control and values similar to studies conducted in the same area 7 years previously. Worrisome is the fact that 66.6% of women of child-bearing age are infected, leading to a high risk of congenital Chagas disease in the future. The present study shows that preventive policies sustained over time are necessary with teams working both on vector control and diagnosis, treatment, and follow-up of patients in endemic areas consisting of specialized health care workers, preferably in the field of pediatrics (AU)

9-Hydroxystearic acid upregulates p21(WAF1) in HT29 cancer cells.

Growing evidence supports the critical role of lipid peroxidation products in the control of cell proliferation. In previous studies we demonstrated the efficient restriction of the proliferation rate in several cell lines resulting from the in vitro treatment with endogenous lipid polar components of cell membranes. Among these, 9-hydroxystearic acid (9-HSA), a primary intermediate of lipid peroxidation, induced a significant arrest in G0/G1 in HT29 colon cancer cells. In response to 9-HSA treatment of HT29 we observed cell growth arrest and increase in p21(WAF1) expression both at the transcriptional and the translational levels. Growth of p21(WAF1)-deleted HCT116 human colon carcinoma cells was not inhibited by 9-HSA. We present evidence that p21(WAF1) is required for 9-HSA mediated growth arrest in human colon carcinoma cells.

The late effects of proton irradiation on cell growth, cell cycle arrest and apoptosis in a human melanoma cell line.

The aim of this work is the in vitro study of the late effects of single proton irradiation on HTB63 human melanoma cell growth, cell cycle and cell death. The experimental conditions were focused on analyzing the effects of irradiation on the periphery of tumour that can be, in clinical practice, close to critical organs. Confluent cell monolayers were irradiated with single doses ranging from 1 - 20 Gy, using proton beams having an energy of 22.6 MeV at the target. Antiproliferative effect of protons, cell cycle analysis and initiation of cell death, were followed 48 hours after irradiation. The inhibition of melanoma cell growth was observed, especially after single application of 12 and 16 Gy. Cell cycle analysis and cell viability have shown the G2/M and G1/G0 arrest of irradiated cells correlating with the increase of the applied dose. The flow cytometric analysis has shown presence of apoptotic nuclei. These data demonstrate that irradiation with protons, under the chosen experimental conditions, have significant effects on melanoma cell growth inhibition being dose dependent, G2/M cell cycle arrest and appearance of apoptotic nuclei, even 48 hours after irradiation. The results obtained may help the understanding of the relationship between cell proliferation, death and cell cycle regulation of melanomas after proton irradiation.

Inhibition of etoposide-induced apoptosis with peptide aldehyde inhibitors of proteasome.

Recent investigations have indicated the involvement of proteasome in programmed cell death. The present studies show that although peptide aldehyde inhibitors of proteasome are by themselves weak inducers of apoptosis, they inhibit the apoptotic effect of the anticancer drug etoposide in rat thymocytes. Acetyl-Leu-Leu-norvalinal (LLnV-al) and other related peptide aldehydes inhibited the increase in caspase activity and DNA fragmentation that followed treatment with etoposide and their effect was related to their potency as proteasome inhibitors. To inhibit etoposide-induced apoptosis, LLnV-al must be present within 3 h of treatment with etoposide, in the same way as the inhibitor of protein synthesis cycloheximide must be. Etoposide caused a rapid accumulation of p53 protein that was not inhibited by LLnV-al, which was also a strong inducer of p53. Peptide aldehydes were also weak activators of caspase activity, suggesting that the same mechanism, i.e. the blocking of proteasome function, both triggers apoptosis and inhibits the effect of etoposide. These results are consistent with a model in which proteasome is selectively involved in the pathway used by etoposide to induce cell suicide.

ATP depletion inhibits glucocorticoid-induced thymocyte apoptosis.

In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some forms of apoptosis, depending on the death trigger, and that in quiescent cells the de-energization of mitochondria is not necessarily linked to apoptosis.

Injury of rat thymocytes caused by exogenous peroxyl radicals in vitro.

The aim of this study was to investigate oxidative cell injury in rat thymocytes under conditions of radical generation exterior to the cell utilizing the thermolabile azocompound 2,2'-azobis(2-amidinopropane) dihydrochloride to generate peroxyl radicals at a constant and reproducible rate. This initiator, being water-soluble and endowed with a positive charge, is suitable for studies on oxidative damage of biomembranes induced in the external water environment. The relationship between cell viability, lipid and thiol oxidation and chain-breaking antioxidant depletion was studied. During the first hour of treatment cell viability decreased slightly, protein sulfhydryl groups were consumed slowly and no significant production of conjugated dienes occurred. After 90 min of incubation, when thymocyte permeability started to increase, the concentration of alpha-tocopherol decreased gradually, significant changes of polyunsaturated fatty acids occurred and a rapid phase of thio oxidation commenced. It can be concluded that, under conditions of an exogenous oxidant challenge, initially the cell membrane provides a physical barrier to the entrance of radicals to the thymocyte. When peroxyl radicals gain access to the membrane and the molecular barrier begins to disorganize, the oxidizable cellular components become susceptible to massive attack.

Hydroxystearic acid effects on CDC2/histone H1 kinase activity in C108 carcinoma cells.

HSA at appropriate concentrations shows cytostatic and/or cytotoxic effects on murine Lewis carcinoma cell line C108. The cytostatic effect is mediated by an arrest in the cell cycle machinery, with accumulation of cells in G2-M. The combination of enzymatic assays, cell cycle kinetics studies and immunoprecipitation shows that HSA causes to a certainty an accumulation of cells in the M phase, while a similar effect in G2 has still to be demonstrated. It also inhibits histone H1 kinase activity up to 95% of that of mitotic cells, having as a direct or indirect target the cdc2 complex.

In vitro effects of hydroxystearic acid on the proliferation of HT29 and I407 cells.

The in vitro effects of hydroxystearic acid on the proliferation of human colon carcinoma cells (HT29) and human embryonic intestine cells (I407) were examined and compared to previous results obtained in murine C108 lung carcinoma cells. The cells were cultured in the presence, or in the absence, of hydroxystearic acid and tested for cell proliferation and viability; the distribution of cells in the cell cycle was evaluated by flow cytometry. Results show that hydroxystearic acid is also an inhibitor of human cell proliferation, and not only of murine C108 cells. Differently from C108 cells, which upon treatment with hydroxystearic acid accumulate in G2-M phases, hydroxystearic acid-treated HT29 cells increase significantly in numbers in G0-G1; I407, embryonic cells used as a control, when treated show only a slight increase in G0-G1.

Effect of hydrostatic pressure and cholesterol depletion on the expression of a tumor-associated antigen.

The molecular events related to the expression of three tumor-associated epitopes, Ca-MOv17, Ca-MOv18 and Ca-MOv19 have been addressed. The epitopes are carried by a 38-kDa glycoprotein (gp38), recently cloned and identified as a human folate-binding protein. They were found to be coexpressed on the surface of the ovarian carcinoma cell line OVCA432, while they are not coordinately expressed on other adenocarcinoma cell lines (IGROV1, HT-29). This lack of coexpression was investigated from a molecular point of view. We studied three carcinoma cell lines, characterized by a different reactivity with the three relevant monoclonal antibodies MOv17, MOv18 and MOv19. The epitope expression was examined after modifying the membrane properties by using hydrostatic pressure and/or the variation of cholesterol content. Measurement of the expression after cell labelling by mAbs was performed by indirect immunofluorescence, using both fluorescence microscopy and flow cytometry. At variance with HT-29 cells, treatment of ovarian carcinoma IGROV1 cells with hydrostatic pressure failed to exert any effect. On IGROV1, instead, cholesterol depletion affected the expression Ca-MOv17, increasing, in the indirect immunofluorescence tests, the proportion of positive cells from 0 to 66 +/- 9%. Moreover, restoring the cholesterol content of the plasma membrane did not reverse the induced epitope expression. In parallel, immunoprecipitation experiments confirmed that, on IGROV1 surface, gp38 was recognized by all three mAbs. The data presented suggest that in IGROV1 cells the selective lacking of the epitope expression is related to the physical state of the plasma membrane. An explanation is provided by the model of membrane microdomains in which epitope expression may be influenced by the cholesterol level of different plasma membrane regions.

Fluorescence and CD studies on the conformation of the gastrin releasing peptide in solution and in the presence of model membranes.

The conformation of the heptacosapeptide hormone, gastrin releasing peptide, has been studied in buffer and in the presence of lipids, using static and dynamic fluorescence and CD. The results obtained show that, in buffer, the hormone exists in a collection of flexible, random coil type conformers, characterized by a beta-turn between residues 14-19. On the other hand, organic solvents can induce some degree of ordered secondary structure in the peptide chain. The marked changes, observed in CD and fluorescence spectra upon addition of lysolecitin micelles and dimyristoylphosphatidylserine vesicles, clearly show that the peptide interacts with lipids, assuming a lipid specific configuration. Interestingly, no significative spectroscopic changes are produced by exposure to dimyristoylphosphatidylcholine vesicles both in the gel and liquid-chrystalline phases, suggesting a requirement for negatively charged lipids during the process of hormone-membrane interaction.

An investigation on lipoperoxidation mechanisms in boar spermatozoa.

Aerobic incubation of washed boar spermatozoa in a heavy metal free medium at 37 degrees C results in a peroxidative breakdown of membrane phospholipids as revealed by malondialdehyde production. In the presence of iron ions, alone and with ascorbate, the amount of malondialdehyde produced increases noticeably. Alkoxy and lipoperoxy radicals are likely involved in these peroxidative processes, while OH. radical does not seem to be essential in the pathway of malondialdehyde formation.

Conformation of bombesin in buffer and in the presence of lysolecithin micelles: NMR, CD, and fluorescence studies.

The conformation of the tetradecapeptide hormone bombesin has been studied in buffer and in the presence of lysolecithin micelles, using static and dynamic fluorescence, CD, and one- and two-dimensional nmr. The results obtained show that in buffer bombesin is present in an extended flexible chain, with no evidence for any ordered secondary structure. A marked change in the CD spectrum is observed changing from buffer to the lipid suspension. Concomitantly, the 1H-nmr spectrum of bombesin, in a D2O lipid dispersion, shows the persistence of resonances due to exchangeable protons and in similar conditions the fluorescence intensity increases. We think therefore that these results strongly support the hypothesis that bombesin interacts with the lipid phase, assuming ordered secondary structure. Finally, the marked dependence of tryptophan fluorescence quantum efficiency and order parameter from the hormone concentration in the presence of lysolecithin but not in buffer leads to the conclusion that bombesin can associate into the lipid matrix.

Conformational flexibility of the hormonal peptide bombesin and its interaction with lipids.

The conformational flexibility of the tetradecapeptide hormone bombesin has been studied using circular dichroism and fluorescence of its single tryptophan residue. The spectral changes observed indicate that the peptide changed from a random flexible coil in solution to a helical structure in lysolecithin micelles and dimyristoylphosphatidylserine vesicles. The tryptophan residue in the lipid complexes was located in a hydrophobic environment. The interaction with lipids was shown to involve both hydrophobic and electrostatic forces.

A potent factor in extracts of the skin of the Australian frog, Pseudophryne coriacea--II. Stimulation of the leech helical muscle in vitro and in vivo.

Extracts of the skin of Pseudophryne coriacea displayed a powerful stimulant action on the leech helical muscle, both in vitro and in vivo. In the isolated dorsal muscle, the extract caused the appearance of vigorous phasic movements, accompanied by rapid increase in tonus, up to intense spasm. Hyoscine, physostigmine, hexamethonium, tubocurarine and alpha-bungarotoxin did not affect the response to the extract; tetrodotoxin, nifedipine and 5-hydroxytryptamine (5-HT) produced a partial blockade. In the intact animal, the extract at first potently stimulated the musculature, evoking the appearance of a succession of incoordinated, spastic movements, with twisting and rolling of the body of the animal. Stimulation was followed by paralysis and death. It is suggested that the pumiliotoxin-like alkaloid of Pseudophryne coriacea, responsible for these effects, acts directly on the helical muscle.

Isolation and enzymatic characterization of the plasmalemma from bovine spermatozoa.

An improved method for the isolation of pure plasma and acrosomal membranes from bull spermatozoa is presented. Plasma membranes were isolated from the spermatozoa of bulls of different breeds, and some enzymatic activity, such as (Na+-K+) ATPase, Ca++ ATPase, Mg++ ATPase, alkaline and acidic phosphatases were assayed. Such enzymatic activity levels differ noticeably from those published by other authors, whose preparations were probably contaminated by other cellular components. Highly statistically significant differences of these activities have been found among the several breeds.

Parallel bioassay of litorin and phyllolitorins on smooth muscle preparations.

Phyllolitorin and Leu8-phyllolitorin, two nonapeptide amides from the skin of the South American frog Phyllomedusa sauvagei, are representatives of a novel bombesin subfamily, characterized by the occurrence in their molecule of a serine residue substituting the usual histidine residue at position 3 from the C-terminus. In parallel bioassay on ten different smooth muscle preparations and rat blood pressure, phyllolitorin and Leu8-phyllolitorin were virtually equiactive, but the two peptides appeared remarkably less potent that litorin in all test preparations, except the rat urinary bladder. The shape of contractions produced by the phyllolitorins and promptness of cessation of their action upon washing seem to indicate a looser binding of these peptides to their receptors and/or a more rapid inactivation, in comparison to litorin.