Surface Activity and Efficiency of Cat-Anionic Surfactant Mixtures.

The surface activity of surfactant mixtures is critically analyzed. Cat-anionic systems, in which two ionic species are mixed in non-stoichiometric ratios, are considered. With respect to the solution behavior, where a substantial decrease of cmc is met compared to the pure components, a moderate effect on surface tension, γ, occurs. Compared to the pure species, the decrease of surface tension for such mixtures is not significant, and no clear dependence on the mole fraction anionic/cationic is met. The surface tension is grossly constant in the whole concentration range. Conversely, the interaction parameter for surfaces, ß surf (calculated by the regular solution theory), is more negative than that for micelle formation, ß mic . This fact suggests that the desolvation of polar heads of the two species at interfaces is largely different. Very presumably, the underlying rationale finds origin in the sizes and solvation of both polar head groups.

Natural Products from Madagascar, Socio-Cultural Usage, and Potential Applications in Advanced Biomedicine: A Concise Review.

Natural products endowed of biological activity represent a primary source of commodities ranging from nutrition to therapeutic agents, as well as cosmetic tools and recreational principles. These natural means have been used by mankind for centuries, if not millennia. They are commonly used all over the world in socio-economical contexts, but are particularly attractive in disadvantaged areas or economically emerging situations all over the world. This is very likely due to the relatively easy recovery of these bioactive principles from the environment, at a low if any cost, as well as ease of administration and the general popular compliance concerning their consumption/ingestion. In this concise review, we focus on some popular bioactive principles of botanical origin which find a wide use in the Madagascan populations. However, due to space limitations, only some of the most common and largely diffused principles in this country are considered. Finally, a possible nanotechnological administration is discussed in the case where a potential therapeutic usage is envisaged.

Interactions and effects of BSA-functionalized single-walled carbon nanotubes on different cell lines.

Functionalized carbon nanotubes (CNTs) have shown great promise in several biomedical contexts, spanning from drug delivery to tissue regeneration. Thanks to their unique size-related properties, single-walled CNTs (SWCNTs) are particularly interesting in these fields. However, their use in nanomedicine requires a clear demonstration of their safety in terms of tissue damage, toxicity and pro-inflammatory response. Thus, a better understanding of the cytotoxicity mechanisms, the cellular interactions and the effects that these materials have on cell survival and on biological membranes is an important first step for an appropriate assessment of their biocompatibility. In this study we show how bovine serum albumin (BSA) is able to generate homogeneous and stable dispersions of SWCNTs (BSA-CNTs), suggesting their possible use in the biomedical field. On the other hand, this study wishes to shed more light on the impact and the interactions of protein-stabilized SWCNTs with two different cell types exploiting multidisciplinary techniques. We show that BSA-CNTs are efficiently taken up by cells. We also attempt to describe the effect that the interaction with cells has on the dielectric characteristics of the plasma membrane and ion flux using electrorotation. We then focus on the BSA-CNTs' acute toxicity using different cellular models. The novel aspect of this work is the evaluation of the membrane alterations that have been poorly investigated to date.

Diameter-dependent release of a cisplatin pro-drug from small and large functionalized carbon nanotubes.

The use of platinum-based chemotherapeutic drugs in cancer therapy still suffers from severe disadvantages, such as lack of appropriate selectivity for tumor tissues and insurgence of multi-drug resistance. Moreover, drug efficacy can be attenuated by several mechanisms such as premature drug inactivation, reduced drug uptake inside cells and increased drug efflux once internalized. The use of functionalized carbon nanotubes (CNTs) as chemotherapeutic drug delivery systems is a promising strategy to overcome such limitations due to their ability to enhance cellular internalization of poorly permeable drugs and thus increase the drug bioavailability at the diseased site, compared to the free drug. Furthermore, the possibility to encapsulate agents in the nanotubes' inner cavity can protect the drug from early inactivation and their external functionalizable surface is useful for selective targeting. In this study, a hydrophobic platinum(IV) complex was encapsulated within the inner space of two different diameter functionalized multi-walled CNTs (Pt(IV)@CNTs). The behavior of the complexes, compared to the free drug, was investigated on both HeLa human cancer cells and RAW 264.7 murine macrophages. Both CNT samples efficiently induced cell death in HeLa cancer cells 72 hours after the end of exposure to CNTs. Although the larger diameter CNTs were more cytotoxic on HeLa cells compared to both the free drug and the smaller diameter nanotubes, the latter allowed a prolonged release of the encapsulated drug, thus increasing its anticancer efficacy. In contrast, both Pt(IV)@CNT constructs were poorly cytotoxic on macrophages and induced negligible cell activation and no pro-inflammatory cytokine production. Both CNT samples were efficiently internalized by the two types of cells, as demonstrated by transmission electron microscopy observations and flow cytometry analysis. Finally, the platinum levels found in the cells after Pt(IV)@CNT exposure demonstrate that they can promote drug accumulation inside cells in comparison with treatment with the free complex. To conclude, our study shows that CNTs are promising nanocarriers to improve the accumulation of a chemotherapeutic drug and its slow release inside tumor cells, by tuning the CNT diameter, without inducing a high inflammatory response.

Delivery of RNA and its intracellular translation into protein mediated by SDS-CTAB vesicles: potential use in nanobiotechnology.

Catanionic vesicles are supramolecular aggregates spontaneously forming in water by electrostatic attraction between two surfactants mixed in nonstoichiometric ratios. The outer surface charges allow adsorption to the biomembrane by electrostatic interactions. The lipoplex thus obtained penetrates the cell by endocytosis or membrane fusion. We examined the possible cytotoxic effects and evaluated the transfection efficiency of one vesicle type as compared to known commercial carriers. We show that the individual components of two different vesicles types, CTAB (cetyltrimethylammonium bromide) and DDAB (didodecyldimethylammonium bromide) are detrimental for cell survival. We also assayed the cytotoxicity of SDS-DDAB vesicles and showed dose and time dependency, with the DDAB component being per se extremely cytotoxic. The transfection efficiency of exogenous RNA mediated by SDS-CTAB increases if vesicles assemble in the presence of the reporter RNA; finally, freezing abrogates the transfection ability. The results of our experimental strategy suggest that catanionic vesicles may be adopted in gene therapy and control of antiproliferative diseases.

Dual nature of translational control by regulatory BC RNAs.

In higher eukaryotes, increasing evidence suggests, gene expression is to a large degree controlled by RNA. Regulatory RNAs have been implicated in the management of neuronal function and plasticity in mammalian brains. However, much of the molecular-mechanistic framework that enables neuronal regulatory RNAs to control gene expression remains poorly understood. Here, we establish molecular mechanisms that underlie the regulatory capacity of neuronal BC RNAs in the translational control of gene expression. We report that regulatory BC RNAs employ a two-pronged approach in translational control. One of two distinct repression mechanisms is mediated by C-loop motifs in BC RNA 3' stem-loop domains. These C-loops bind to eIF4B and prevent the factor's interaction with 18S rRNA of the small ribosomal subunit. In the second mechanism, the central A-rich domains of BC RNAs target eIF4A, specifically inhibiting its RNA helicase activity. Thus, BC RNAs repress translation initiation in a bimodal mechanistic approach. As BC RNA functionality has evolved independently in rodent and primate lineages, our data suggest that BC RNA translational control was necessitated and implemented during mammalian phylogenetic development of complex neural systems.

Biological activity of SDS-CTAB cat-anionic vesicles in cultured cells and assessment of their cytotoxicity ending in apoptosis.

SDS-CTAB cat-anionic vesicles are supramolecular aggregates forming complexes with biopolymers and enter the cells via membrane fusion or endocytosis. Different applicative areas exist: gene therapy, drug delivery and nanotechnology. We previously examined the absorption/release of biopolymers from vesicles in solution. Here we evaluate their cytotoxicity in cultured cells; to this end we characterized the vesicles and analyzed their biological effects at cellular and molecular level. At low concentration these vesicles have scarce consequences on normal cell growth; at higher dosage they activate apoptotic death processes, due to membrane damage. In conclusion, the use of these particles in nano-biotechnology represents an actual possibility.

Regeneration and DNA demethylation do not trigger PDX-1 expression in rat hepatocytes.

AIM: To explore the possibility that PDX-1 gene is reactivated as a consequence of molecular events that occur during liver regeneration. METHODS: Rat hepatocytes were maintained in DMEM-F12, 10% fetal bovine serum (FBS), penicillin/streptomycin and geneticin when applicable. Rat insulinoma RIN 1046-38 cells were maintained in M-199-10% FBS and penicillin/streptomycin. The final concentration of glucose was 11.1 mmol/L. During regeneration, lateral and medial liver lobes of adult male Wistar rats were surgically removed, with up 70% loss of liver mass. In methylation experiments, 5-aza-deoxycytidine (5-aza-dC) was used. Primer3 software was used for polymerase chain reaction (PCR). Quantitative real time PCR (qRT-PCR) was performed using SYBR Green technology; primers were designed by Beacon Designer 6 software. Western blotting and SDS-PAGE were performed according to standard procedures. Antibodies were purchased from commercial suppliers. RESULTS: We explored the possibility that liver regeneration could trigger PDX-1 expression, and hence insulin production. Twenty-four hours after surgical liver removal, regeneration was active as demonstrated by the increased proliferating cell nuclear antigen; however, all the other checked genes (involved in insulin gene expression): PC-1, Ngn3, NeuroD1, Btc, PDX-1 and Ins-1, were not related to the molecular events caused by this process. The only marker detected in regenerating liver was E47: a transcription factor of the the basic helix-loop-helix family known to be expressed ubiquitously in mammalian cells. In the rat pancreas, almost all of the tested genes were expressed as shown by RT-PCR, except for Ngn3, which was silenced 2 d after birth. Therefore, the molecular events in liver regeneration are not sufficient to promote PDX-1 expression. DNA methylation is a known mechanism to achieve stable repression of gene expression in mammals: Hxk 2 gene is silenced through this mechanism in normal hepatocytes. The administration of 5-aza-dC to cultured cells is in fact able to upregulate Hxk 2 mRNA. We investigated whether PDX-1 silencing in liver cells could be exerted through methylation of CpG islands in both the promoter and the gene coding regions. The results show that the drug increased the expression level of the Hxk 2 control gene but failed to rescue the expression of PDX-1, thus DNA demethylation is not sufficient to override repression of the PDX-1 gene. CONCLUSION: During liver regeneration, PDX-1 gene is not reactivated. Demethylation does not de-repress PDX-1 gene expression. Therefore gene silencing is not achieved through this epigenetic mechanism.

The synthetic inhibitor of fibroblast growth factor receptor PD166866 controls negatively the growth of tumor cells in culture.

BACKGROUND: Many experimental data evidence that over-expression of various growth factors cause disorders in cell proliferation. The role of the Fibroblast Growth Factors (FGF) in growth control is indisputable: in particular, FGF1 and its tyrosine kinase receptor (FGFR1) act through a very complex network of mechanisms and pathways. In this work we have evaluated the antiproliferative activity effect of PD166866, a synthetic molecule inhibiting the tyrosin kinase action of FGFR1. METHODS: Cells were routinely grown in Dulbecco Modified Eagle's medium supplemented with newborn serum and a penicillin-streptomycin mixture.Cell viability was evaluated by Mosmann assay and by trypan blue staining. DNA damage was assessed by in situ fluorescent staining with Terminal Deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL assay).Assessment of oxidative stress at membrane level was measured by quantitative analysis of the intra-cellular formation of malonyl-dialdheyde (MDA) deriving from the decomposition of poly-unsaturated fatty acids.The expression of Poly-ADP-Ribose-Polymerase (PARP), consequent to DNA fragmentation, was evidenced by immuno-histochemistry utilizing an antibody directed against an N-terminal fragment of the enzyme. RESULTS: The bioactivity of the drug was investigated on Hela cells. Cytoxicity was assessed by the Mosmann assay and by vital staining with trypan blue. The target of the molecule is most likely the cell membrane as shown by the significant increase of the intracellular concentration of malonyl-dihaldheyde. The increase of this compound, as a consequence of the treatment with PD166866, is suggestive of membrane lipoperoxidation. The TUNEL assay gave a qualitative, though clear, indication of DNA damage. Furthermore we demonstrate intracellular accumulation of poly-ADP-ribose polymerase I. This enzyme is a sensor of nicks on the DNA strands and this supports the idea that treatment with the drug induces cell death. CONCLUSIONS: Data presented in this work show that PD166866 has clear antiproliferative effects. The negative control of cell proliferation may be exerted through the activation of the apoptotic pathway. The results of experiments addressing this specific point, such as: evaluation of DNA damage, lipoperoxidation of the cell membrane and increase of expression of PARP, an enzyme directly involved in DNA repair. Results suggest that cells exposed to PD16866 undergo apoptosis. However, concomitant modes of cell death cannot be ruled out. The possible use of this drug for therapeutic purposes is discussed.

Resveratrol exhibits a strong cytotoxic activity in cultured cells and has an antiviral action against polyomavirus: potential clinical use.

BACKGROUND: Resveratrol is a non flavonoid polyphenol compound present in many plants and fruits and, at especially high concentrations, in the grape berries of Vitis vinifera. This compound has a strong bioactivity and its cytoprotective action has been demonstrated, however at high concentrations the drug exhibits also an effective anti-proliferative action. We recently showed its ability to abolish the effects of oxidative stress in cultured cells. In this work we assayed the bioactivity of resveratrol as antiproliferative and antiviral drug in cultured fibroblasts. Studies by other Authors showed that this natural compound inhibits the proliferation of different viruses such as herpes simplex, varicella-zoster and influenza A. The results presented here show an evident toxic activity of the drug at high concentrations, on the other hand at sub-cytotoxic concentrations, resveratrol can effectively inhibit the synthesis of polyomavirus DNA. A possible interpretation is that, due to the damage caused by resveratrol to the plasma membrane, the transfer of the virus from the endoplasmic reticulum to the nucleus, may be hindered thus inhibiting the production of viral DNA. METHODS: The mouse fibroblast line 3T6 and the human tumor line HL60 were used throughout the work. Cell viability and vital cell count were assessed respectively, by the MTT assay and Trypan Blue staining. Cytotoxic properties and evaluation of viral DNA production by agarose gel electrophoresis were performed according to standard protocols. RESULTS: Our results show a clear dose dependent both cytotoxic and antiviral effect of resveratrol respectively at high and low concentrations. The cytotoxic action is exerted towards a stabilized cell-line (3T6) as well as a tumor-line (HL60). Furthermore the antiviral action is evident after the phase of virion entry, therefore data suggest that the drug acts during the synthesis of the viral progeny DNA. CONCLUSION: Resveratrol is cytotoxic and inhibits, in a dose dependent fashion, the synthesis of polyomavirus DNA in the infected cell. Furthermore, this inhibition is observed at non cytotoxic concentrations of the drug. Our data imply that cyto-toxicity may be attributed to the membrane damage caused by the drug and that the transfer of polyomavirus from the endoplasmic reticulum to the cytoplasm may be hindered. In conclusion, the cytotoxic and antiviral properties of resveratrol make it a potential candidate for the clinical control of proliferative as well as viral pathologies.

Alterations of the plasma membrane caused by murine polyomavirus proliferation: an electrorotation study.

In this report we investigate the alterations of the dielectric properties of the plasma membrane caused by the infection of cultured fibroblasts with murine polyomavirus. The approach consists in a well-established dielectric spectroscopy technique, electrorotation, which has been successfully used in our laboratory to study the alterations of the plasma membrane of cells exposed to various forms of stress. The response to viral proliferation was time dependent as shown by evaluation of the de novo synthesis of viral DNA. This response was paralleled by gradual damage of the membrane evidenced by alteration of the dielectric parameters, specific capacitance and conductance. The electrorotation results show a reduced effect on the dielectric properties of the membrane when infection is carried out in the presence of a natural oil (MEX). In this case a drastic reduction in viral DNA synthesis was also monitored, thus indicating an antiviral action of this product.

Overexpression of the Pdx-1 homeodomain transcription factor impairs glucose metabolism in cultured rat hepatocytes.

The Pdx-1 transcription factor plays crucial functions both during pancreas development and in the adult beta cells. Previous studies have indicated that ectopic Pdx-1 expression in liver or intestinal primary and immortalized cells is sufficient to promote activation of insulin gene expression. This work is focused on the molecular and physiological consequences of Pdx-1 overexpression in liver cells. We present evidence that Pdx-1 affects the level of expression of one of the four mammalian hexokinase isozymes. These are glucose phosphorylating enzymes involved in essential cellular functions such as glucose sensing, metabolic energy production and apoptosis. Specifically, our data show that over-expression of Pdx-1 in cultured hepatocytes is able to repress the expression of hexokinase 2 (Hxk 2) and the phenomenon is mediated via binding of Pdx-1 to a specific sequence on the Hxk 2 gene promoter. As a consequence, liver cells over-expressing Pdx-1 present interesting alterations concerning glucose metabolism.

Dynamics of DNA adsorption on and release from SDS-DDAB cat-anionic vesicles: a multitechnique study.

DNA adsorption and release from cat-anionic vesicles made of sodium dodecylsulfate-dodecyldimethylammonium bromide (SDS-DDAB) in nonstoichiometric amounts was investigated by different electrochemical, spectroscopic, and biomolecular strategies. The characterization of the vesicular system was performed by dynamic light scattering, which allowed estimating both its size and distribution function(s). The interaction dynamics was followed by dielectric spectroscopy and zeta-potential, as well as by agarose gel electrophoresis, AGE. Also, circular dichroism, CD, measurements were carried out, to ascertain possible structural rearrangements of DNA, consequent to the interactions with the cat-anionic vesicles. CD demonstrates that vesicle-bound DNA retains its native conformation. The results obtained by the aforementioned techniques are consistent and indicate that binding saturation is obtained at a [DNA/vesicles] charge ratio close to 0.8, considering only the excess surface charges on the vesicles. This result is apparently in contradiction with a purely electrostatic approach and is tentatively ascribed to the distance between charges on the biopolymer and the vesicle surface, respectively. A possible interpretation is discussed. The nucleic acid can be completely retrieved from the vesicles upon addition of adequate amounts of SDS, which is the defective surfactant in the vesicular system. Precipitation of the poorly soluble SD-DDA salt results in an almost complete release of DNA.

Differential cytotoxicity of MEX: a component of Neem oil whose action is exerted at the cell membrane level.

Neem oil is obtained from the seeds of the tree Azadirachta indica. Its chemical composition is very complex, being rich in terpenoids and limonoids, as well as volatile sulphur modified compounds. This work focused on the evaluation of a component of the whole Neem oil obtained by methanolic extraction and defined as MEX. Cytotoxicity was assessed on two different cell populations: a stabilized murine fibroblast line (3T6) and a tumor cell line (HeLa). The data presented here suggest a differential sensitivity of these two populations, the tumor line exhibiting a significantly higher sensitivity to MEX. The data strongly suggest that its toxic target is the cell membrane. In addition the results presented here imply that MEX may contain one or more agents that could find a potential use in anti-proliferative therapy.

Charge redistribution in adenosylribosyl transferase caused by substitution of a single amino acid residue.

Dielectric measurements in the frequency range 10(5)-10(8) Hz were performed on wild-type (wt) adenosylribosyl transferase and a mutant enzyme. The analysis of the dielectric relaxation curve allowed the estimation of the hydrodynamic radius and of the electric dipole moment. The first parameter remained unchanged in wt and mutant protein. The dipole moment of the mutant, however, was significantly increased. Implications on the electrostatic interactions between enzyme and substrate are discussed.

Metabolic alterations in cultured mouse fibroblasts induced by an inhibitor of the tyrosine kinase receptor Fibroblast Growth Factor Receptor 1.

Proton nuclear magnetic resonance (NMR) spectroscopy was used to identify and quantify the metabolites present in cultured mouse fibroblast cells 3T6 in their native state and after treatment with PD166866, an inhibitor of the fibroblast growth factor receptor. Cell extracts were prepared according to the Bligh-Dyer protocol which prevents artifacts deriving from the chemical demolition of macromolecules. Also the growth medium was subjected to the same extraction procedure. The NMR approach made possible the identification and quantification of about 40 different metabolites at nanomoles/mg of protein level: the biological relevance of the variation of some metabolite levels is discussed. Our experimental procedure offers a prospective method for the evaluation of variations of the metabolic profile deriving from different biochemical treatments of these cells.

A biophysical investigation on the binding and controlled DNA release in a cetyltrimethylammonium bromide-sodium octyl sulfate cat-anionic vesicle system.

The interactions between cat-anionic (an acronym indicating surfactant aggregates (micelles and vesicles) formed upon mixing cationic and anionic surfactants in nonstoichiometric amounts) vesicles and DNA have been the subject of intensive studies because of their potential applications in biomedicine. Here we report on the interactions between DNA and cetyltrimethylammonium bromide (CTAB)-sodium octyl sulfate (SOS) cat-anionic vesicles. The study was performed by combining dielectric relaxation spectroscopy, circular dichroism, dynamic light scattering, ion conductivity, and molecular biology techniques. DNA is added to positively charged vesicles until complete charge neutralization of the complex and formation of lipoplexes. This occurs when the mole ratio between the phosphate groups of DNA and positive charges on the vesicle is about 1.8. Above this threshold the nucleic acid in excess remains free in solution. This very interesting new result shows that anionic surfactants are not expelled upon saturation, and therefore, no formation of micelles occurs. Furthermore, vesicle-bound DNA can be released in its native form, as confirmed by dielectric spectroscopy and circular dichroism measurements. The nucleic acid is released upon addition of SOS, which competes with the phosphate groups of the DNA: this results in the demolition of the CTAB-SOS cat-anionic vesicles. These results indicate the possibility of a controlled DNA release and might be of interest in biomedicine.

Dielectric properties of the plasma membrane of cultured murine fibroblasts treated with a nonterpenoid extract of Azadirachta indica seeds.

Neem oil is a natural product obtained from the seeds of the tree Azadirachta indica. In this report, we investigate the alterations of the biophysical properties of the plasma membrane caused by treatment with the nonterpenoid fraction of neem oil that we defined as methanolic extract (MEX). The dose-response effect was evaluated and a MEX-dependent cytoxicity evidenced. The effect of MEX on the plasma membrane was studied by a well-established dielectric spectroscopy technique: electrorotation, which allows single-cell analysis. Our results show a structural/functional alteration of the plasma membrane with an evident increase of specific capacitance and conductance. The biological implications of this effect are discussed.

Degenerative and apoptotic events at retinal and optic nerve level after experimental induction of ocular hypertension.

Ocular hypertension is a symptom of a glaucomatous condition characterized by a severe vision decrease. Blindness caused by the apoptotic death of the retinal ganglion cells and of the astrocytes of the optic nerve may eventually result. Experimental hypertension was induced by inoculation of methylcellulose in the anterior chamber. Chromatin staining, TUNEL assay, and inter-nucleosomal DNA fragmentation observed in retina and optic nerve strongly suggest that hypertension causes apoptosis. Immunolocalization of the fibrillary acidic glial protein, specific of cell stress, and caspase-3 in the same tissues, further support this mode of cell death. Activation of the ubiquitin dependent proteolytic system was also observed. Protection from apoptosis exerted by administration of the peroxide scavenger trolox, suggests that the apoptotic pathway is activated by an oxidative stress. The data presented here show that the experimental hypertensive insult induces degenerative and apoptotic events comparable to those observed in human glaucoma.

Cytotoxic and antiproliferative effects induced by a non terpenoid polar extract of A. indica seeds on 3T6 murine fibroblasts in culture.

Neem oil is a natural product obtained from the seeds of the tree Azadirachta indica. Its composition is very complex and the oil exhibits a number of biological activities. The most studied component is the terpenoid azadirachtin which is used for its insecticidal and putative antimicrobial properties. In this report we investigate the biological activity of partially purified components of the oil obtained from A. indica. We show that the semi-purified fractions have moderate to strong cytotoxicity. However, this is not attributable to azadirachtin but to other active compounds present in the mixture. Each fraction was further purified by appropriate extraction procedures and we observed a differential cytotoxicity in the various sub-fractions. This led us to investigate the mode of cell death. After treatment with the oil fractions we observed positivity to TUNEL staining and extensive internucleosomal DNA degradation both indicating apoptotic death. The anti-proliferative properties of the neem oil-derived compounds were also assayed by evaluation of the nuclear PCNA levels (Proliferating Cell Nuclear Antigen). PCNA is significantly reduced in cells treated with a specific fraction of neem oil. Finally, our results strongly suggest a possible involvement of the mitochondrial pathway in the apoptotic death.