Cationic liposomes formulated with DMPC and a gemini surfactant traverse the cell membrane without causing a significant bio-damage.

Cationic liposomes have been intensively studied both in basic and applied research because of their promising potential as non-viral molecular vehicles. This work was aimed to gain more information on the interactions between the plasmamembrane and liposomes formed by a natural phospholipid and a cationic surfactant of the gemini family. The present work was conducted with the synergistic use of diverse experimental approaches: electro-rotation measurements, atomic force microscopy, ζ-potential measurements, laser scanning confocal microscopy and biomolecular/cellular techniques. Electro-rotation measurements pointed out that the interaction of cationic liposomes with the cell membrane alters significantly its dielectric and geometric parameters. This alteration, being accompanied by significant changes of the membrane surface roughness as measured by atomic force microscopy, suggests that the interaction with the liposomes causes locally substantial modifications to the structure and morphology of the cell membrane. However, the results of electrophoretic mobility (ζ-potential) experiments show that upon the interaction the electric charge exposed on the cell surface does not vary significantly, pointing out that the simple adhesion on the cell surface of the cationic liposomes or their fusion with the membrane is to be ruled out. As a matter of fact, confocal microscopy images directly demonstrated the penetration of the liposomes inside the cell and their diffusion within the cytoplasm. Electro-rotation experiments performed in the presence of endocytosis inhibitors suggest that the internalization is mediated by, at least, one specific pathway. Noteworthy, the liposome uptake by the cell does not cause a significant biological damage.

The cell membrane is the main target of resveratrol as shown by interdisciplinary biomolecular/cellular and biophysical approaches.

One of the research lines developed in our laboratory is focused on the study of the bioactivity of natural substances. Resveratrol (RV) is a polyphenol nonflavonoid compound present in a number of plant species but mainly in the berries of the red grape Vitis vinifera. The powerful antioxidant action of this molecule is well documented. In this work we evaluated the effects of this substance by adopting diverse experimental strategies. In particular, we studied the effects on cell vitality and cycle by MTT and cytofluorimetric assays. In addition, we explored the action of RV on the cell membrane by a well-consolidated biophysical approach: electrorotation. This technique allows assessment of the structure/function of the cell membrane. The results presented here demonstrate that RV shows a modest effect on the biological properties of the cell in terms of cytotoxicity and cell cycle alterations. On the contrary, a significant effect on the membrane structure/function was observed, consisting of an enhanced intramembrane ion transport. The implications and interpretation of these membrane alterations are discussed.

Interactions of DMPC and DMPC/gemini liposomes with the cell membrane investigated by electrorotation.

The electrorotation technique was utilized to investigate the interactions between a mouse fibroblast cell line and zwitterionic liposomes formed by a natural phospholipid or cationic liposomes formulated with the same phospholipid and a cationic gemini surfactant. The application of this technique allowed an accurate characterization of the passive dielectric behavior of the plasma membrane by the determination of its specific capacitance and conductance. Changes of these parameters, upon interaction with the liposomes, are related to variations in the structure and or in the transport properties of the membrane. Cells were exposed to both types of liposomes for 1 or 4h. Electrorotation data show a dramatic reduction of the dielectric parameters of the plasma membrane after one hour treatment. After 4h of treatment the effects are still observed only in the case of the cationic liposomes. Surprisingly, these same treatments did not cause a relevant biological damage as assessed by standard viability tests. A detailed discussion to rationalize this phenomenon is presented.

Carnitine reduces the lipoperoxidative damage of the membrane and apoptosis after induction of cell stress in experimental glaucoma.

The pathological damage caused by glaucoma is associated to a high intraocular pressure. The ocular hypertone is most likely due to a defective efflux of aqueous humor from the anterior chamber of the eye. Ocular hypertension causes apoptotic death of retinal ganglion cells and overexpression of molecular markers typical of cell stress response and apoptosis. In this work, we report on the neuroprotective, antiapoptotic and antioxidant action of a natural substance, -carnitine. This compound is known for its ability to improve the mitochondrial performance. We analyze a number of cellular and molecular markers, typical of ocular hypertension and, in general, of the cell stress response. In particular, L-carnitine reduces the expression of glial fibrillary acidic protein, inducible nitric oxide synthase, ubiquitin and caspase 3 typical markers of cell stress. In addition, the morphological analysis of the optic nerve evidenced a reduction of the pathological excavation of the optic disk. This experimental hypertone protocol induces a severe lipoperoxidation, which is significantly reduced by L-carnitine. The overall interpretation is that mortality of the retinal cells is due to membrane damage.

Reduction of cell proliferation induced by PD166866: an inhibitor of the basic fibroblast growth factor.

UNLABELLED: Cell proliferation control plays a key role in tumor development. The basic Fibroblast Growth Factor (bFGF), as well as other growth factors, is involved in several pathologies characterized by dysregulation of cell proliferation. In the present work the effects of PD166866, a very potent and selective tyrosine kinase inhibitor were evaluated. Cultured murine fibroblasts (the cell line 3T6) were used to assess the FGFR-1 inhibition mediated by PD166866. Evaluation of cell viability and molecular biology techniques were adopted. PD166866 controls negatively the bFGF/FGFR-1 system thus promoting a significant reduction of cell proliferation and loss of viability in 3T6 cells. The drug possibly controls proliferation via induction of apoptosis as evidenced by a relevant chromatin degradation. CONCLUSION: This study demonstrated that PD166866 might be used in the control of fibrotic proliferative diseases, as well as in other tumor pathologies.

The diimide drug PIPER has a cytotoxic dose-dependent effect in vitro and inhibits telomere elongation in HELA cells.

It is known that, in vitro, PIPER (N,N'-bis [2-(1-piperidino)ethyl]-3,4,9,10-tetracarboxylic diimide) induces the formation of the Hoogsteen quadruplex structure in telomere DNA, thus inhibiting the polymerisation of telomeric repeats. Since the action of PIPER in vivo has been scarcely investigated, this study was addressed to gain some insight into the effects of this drug on cultured HeLa cells. Vital staining with erythrosine, performed on cells exposed to different PIPER concentrations (from 1 to 50 microM), showed that the drug exerts a dose-dependent cytotoxic effect, clearly evident after a short-term (24 h) treatment. This early cytotoxic effect of PIPER on cultured HeLa cells was confirmed by a spectrophotometric/colorimetric method employing methylthiazoletetrazolium (Mossmann assay). Hematoxylin/eosin staining of cells treated with PIPER for 24 h showed a nuclear condensation and a cytoplasmic vacuolisation, very pronounced at higher drug concentrations. These pictures suggest that PIPER-induced cell death might be of the apoptotic type. Finally, the anti-telomerase activity of PIPER was monitored by TRAP assay, performed on HeLa cell nuclear extracts treated with increasing drug concentrations. It was found that some inhibition of telomerase is apparent even at low concentrations, while at the highest concentration the enzyme is completely inhibited. These results indicate that the cytotoxic power of PIPER is possibly related to its antitelomeric effect.

Presence and incidence of DNA sequences of human polyomaviruses BKV and JCV in colorectal tumor tissues.

The human polyomaviruses JCV and BKV are widespread within population, as shown by serological studies. However, exposure to these viruses does not seem to have pathological consequences in immunocompetent individuals, while in immunocompromised or immunosuppressed patients, polyomaviruses can be activated, giving rise to serious pathologies. Viral DNA sequences were also found in cells from a number of human tumors of mesothelial origin, suggesting that activation of BKV and JCV, taking place in genetically predisposed and/or in immunodepressed individuals, might be involved in the mechanisms of tumor transformation. In this study, samples obtained from 18 patients with colon rectal carcinoma were probed for the presence of JCV and BKV by three different techniques: Southern blot, PCR and in situ hybridization. Our results demonstrate that viral DNA sequences were present in 16 out of the 18 cases considered (88.9%). In the large majority of cases, viruses were detected both in the tumor mass and in the surrounding healthy tissues. Lymphocytes in the investigated areas were also found to be infected by polyomaviruses. These data indicate, for the first time, a possible involvement of polyomaviruses in the pathogenesis of tumors of endothelial origin, like the human colon rectal carcinoma.

[Molecular epidemiologic analysis of the levels of metalloproteases and cyclooxygenase-2 in colorectal cancer]. / Analisi epidemiologico molecolare dei livelli di metalloproteasi e cicloossigenasi-2 nel cancro del colon/retto.

The aim of this work is the investigation of the level of expression of the vascular endothelial growth factor (VEGF) and metalloproteases (such as Cox-2) in the colon rectal cancer. The final goal is the correlation with liver metastases. Molecular biology approaches will be adopted: in particular: RT-PCR, quantitative real time PCR with fluorescent probes, immunolocalizations and Western blotting This is a proposal of research. The work is under development and results and conclusions cannot be at the moment anticipated. However, in recent publication we found human polyomavirus DNA sequences in colorectal tumors.

Variations of telomerase activity in cultured mouse fibroblasts upon proliferation of polyomavirus.

Telomerase plays a central role in various biological phenomena such as cell differentiation and proliferation, apoptosis, malignant transformation and virus infection, for instance HIV and papillomavirus. In addition, it has recently been shown that, in human fibroblasts transformed by monkey polyomavirus SV40, telomeres became stabilized as a consequence of telomerase activation. However, no information exists on the effects of acute infection by murine polyomavirus on the telomeres maintenance and telomerase activity in the host cell. In this paper we report on a differential activity of telomerase in productively infected cells. The results showed a decreased activity of the enzyme as assessed by the TRAP assay. The decrease had already occurred at a non-lytic time of infection and was observed both after infection and naked DNA transfection. Therefore nuclear decapsidation is not involved in the determination of the phenomenon that is attributed to the proliferation of the virus.

Molecular characterization and action of usnic acid: a drug that inhibits proliferation of mouse polyomavirus in vitro and whose main target is RNA transcription.

Usnic acid is a normal component of lichen cells. This natural compound has shown different biological and physiological activities that might have a great relevance in pharmacology and clinics. For instance, usnic acid is known for its antibacterial and antiparasitic action. Also, the drug has a potential interest in cancer therapy because of its antimitotic and antiproliferative action. The molecular structure of usnic acid has been validated and further explored in this investigation. Many biological properties of this drug are known; however its potential antiviral action has not yet been evaluated. In this paper, we demonstrate that usnic acid is a potent inhibitor of the proliferation of mouse polyomavirus. Its action is not exerted at the level of virion entry into the host cell. Moreover, the abolition of viral DNA replication is an indirect consequence of the drastic inhibition of RNA transcription.

Ubiquitin dependent proteolysis is activated in apoptotic fibroblasts in culture.

The ubiquitin mediated pathway constitutes an early response in cultured cells where apoptosis, assessed by internucleosomal specific DNA fragmentation, was induced by serum withdrawal. Data demonstrate that nuclear ubiquitin proteolytic system, but not cytoplasmic, is activated. This activation is paralleled by a substantial chromatin de-condensation. We suggest that chromatin relaxation is causative of the fragmentation since it exposes the DNA to nucleolytic attack. Finally, maintenance of homeostasis and induction of apoptosis seem to undergo a parallel contemporary pathway with a possible mutual feedback.

A murine cell culture model for post-trabeculectomy anfibrotic treatment: Induction of apoptosis by Cyclosporin.

PURPOSE: Experimental trials aimed at the research of selective antifibrotic agents are under development for the alternative treatment of glaucoma patients who are usually considered high-risk post-surgical individuals after trabeculectomy. Authors present here an in vitro model system for the treatment of post-trabeculectomy patients. The study is aimed at the evaluation of different drugs in a mouse fibroblast model. METHODS: The antifibrotic activity of Cyclosporin A, Interferon 2alpha, 5-Fluorouracyl was investigated on 3T6 cells in culture. Cell viability and proliferation was assessed after drug treatment. Molecular analysis of DNA degradation was evaluated by means of radioactive labeling and gel electrophoresis. RESULTS: The three drugs were shown to affect cell proliferation and viability in a differential fashion. However, only Cyclosporin A was able to control cell proliferation, inducing apoptosis. This phenomenon was reduced by supplementation of trolox, a compound known to inhibit programmed cell death. These results strongly suggest that this model system might be useful as a test of pharmacological functionality. CONCLUSION: A rapid and efficient model system is described for the assessment of cell viability and proliferation after treatment with agents of potential pharmacological use. Cyclosporin A induces a significant apoptosis. This is important for the negative control of fibrotic degeneration in post-trabeculectomy that is required for successful surgery in glaucoma patients. Therefore, Cyclosporin A might become a clinically interesting drug for the antifibrotic treatment of post-trabeculectomy.

Intrinsic structural differences between "tight couples" and Kaltschmidt-Wittmann ribosomes evidenced by dielectric spectroscopy and scanning microcalorimetry.

Measurements of dielectric spectroscopy (DS) and microcalorimetry (differential scanning calorimetry (DSC)) of Escherichia coli 70S, 50S and 30S were performed on particles prepared according either to the "classical" twice NH(4)Cl-washed ribosomes, also known as loose couples (LC), or to the "tight couples" preparative protocol (TC). Results show that 70S particles prepared according to the two different protocols exhibit different structural properties. Two subsequent relaxation processes occur in both samples as measured by DS. However, in LC ribosomes the first one is shifted towards a lower frequency with a higher dielectric increment. This is suggestive of a more extensive exposure of RNA to the solvent and of an overall more relaxed structure. The smaller LC subunit exhibits only one relaxation while the TC 30S shows two dielectric dispersions as well as 70S. No substantial differences were evidenced in either 50S species. Two typical melting peaks were observed by DSC both in LC and TC 70S as well as in 50S. Thermograms obtained from the TC 30S show a single well structured peak while LC particles produce a large unstructured curve. On the basis of these results we conclude that TC 70S particles are more compact than LC ribosomes and that in the former ones the rRNA is less exposed to the solvent phase. Furthermore 30S particles obtained from TC show a more stable structure with respect to LC 30S. We conclude that the 30S subunit gives a major contribution to the compact character of the whole TC 70S. These differences might be related to the intrinsic and well documented functional difference between the two ribosome species.

Critical analysis of the impedance method for the evaluation of permittivity and conductivity of the plasma membrane.

We report a critical analysis of a typical method of dielectric spectroscopy consisting in impedance measurements as a function of frequency. Experimental data were obtained by measuring impedance on human erythrocyte suspensions. Since these cells do not have a nucleus they represent an ideal material for the application of the well established single shell model. This allows the evaluation of permittivity and conductivity of the plasma membrane. We discuss the influence on the reliability of results of parameters such as fractional volume, average dimensions and membrane thickness of cells.

Instability of three-dimensional structures in ribosomal cores evidenced by microcalorimetric studies.

In this paper we show a microcalorimetric investigation carried out on the so-called cores, i.e. ribosomes deprived of select proteins by LiCl treatment. Thermal degradation of native ribosomes gives rise to two thermal transitions occurring at different temperatures. In the cores the high temperature peak persists even after treatment at very high ion strength (2 M LiCl). This strongly suggests the existence of a very stable structure that was previously observed also in particles treated with agents that hydrolyze the RNA moiety. The low temperature peak gradually but dramatically decreases even though it never disappears completely. This indicates that the treatment to obtain ribosomal cores does not cause complete unfolding of the particle but only the destabilization of a structural three-dimensional domain present in native ribosomes. These data are discussed in the light of previous results obtained by dielectric spectroscopy and microcalorimetric studies on ribosomal particles.

Effect of different whole body hyperthermic sessions on the heat shock response in mice liver and brain.

We examined by Western blots the effect of variations of the heating sessions, such as duration and intensity on the following aspects: 70-kDa heat shock protein (HSP70) and HSP72 induction. Protein ubiquitination PLCgamma , PKCepsilon and PKCalpha levels in murine liver and brain were also studied. Results demonstrated that maximal induction of HSP72 was obtained after heat shock at 43.5 degrees C in both organs. Preconditioning at lower temperatures (either acclimation to 39 degrees C or induction of thermotolerance to 43.5 degrees C with a single exposure to 39 degrees C) attenuated the heat shock response. Hepatic HSP72 induction was elicited only as a consequence of hyperthermia since either fasting or restraint were unable to trigger its synthesis. On the contrary, a ubiquitination decrease of a 31 kDa protein was obtained both after hyperthermia and fasting This indicates that the latter is a more generic response of hepatic cells to noxious stimuli. Analysis of the above mentioned enzymes showed that in liver of naive mice PKCalpha is barely present while PKCepsilon is quite abundant. All hyperthermic treatments caused a general decrease of the latter, except for the heat shock at 43.5 degrees C that caused an increase. PLCgamma decreased after all heating sessions. It is known that hyperthermia in the range of 41-45 degrees C induces apoptotic death in many cell types. Therefore we analyzed the presence of the typical apoptotic DNA ladder. Our data strongly suggest that both hyperthermia and restraint induce necrosis in liver while apoptosis and necrosis become evident in brain. All these effects are still present 24 h from the last heating session: This indicates that in vivo, hyperthermia produces long term modifications of the hepatic cell.

The ionophore monensin inhibits mouse polyomavirus DNA replication and destabilizes viral early mRNAs.

Monensin is a ionophore compound with different biological activities. It raises the intralysosomal pH, it binds the plasma membranes particularly at the level of the cisternal system of the Golgi apparatus. It causes imbalance in the intramembrane ion traffic and inhibits export of secretory proteins at membrane level. Monensin blocks endocytosis and therefore impedes entry of toxic molecules. The drug also inhibits viral proliferation of RNA and DNA viruses such as vesicular stomatitis, influenza and human polyomaviruses. In this report we show that monensin effectively abolishes viral DNA replication of mouse polyomavirus. Results show that the half life of viral early mRNAs is significantly reduced in the presence of the drug. Therefore we suggest that the reduction of viral DNA synthesis is a consequence of the reduced intranuclear pool of viral early antigens.

Structural stability of ribosomes subjected to RNase treatment evidenced by dielectric spectroscopy and differential scanning microcalorimetry.

Previous studies from our laboratory demonstrated the existence of at least two levels of structural complexity in E. coli 70S ribosomes. Ribosomal RNA seems to be principally involved in the overall stability of these structures. In this paper we present an investigation of ribosomes subjected to treatment with RNase. The study is based on both differential scanning microcalorimetry and dielectric spectroscopy. In the thermograms obtained on treated ribosomes only the low temperature peak of the two typical denaturation events observed in native ribosomes, is promptly eliminated by the enzyme treatment. Dielectric spectroscopy measurements carried out on the same samples indicate an alteration of the dielectric behavior previously shown to consist of two subsequent relaxation processes. In fact, only the low frequency relaxation is affected by the treatment. The second one, observed at higher frequency, remains unaltered. The same effect on the dielectric parameters is observed if the ribosome particles are heated and then cooled prior to measurement. These results are consistent with the idea that two different structures are present within the ribosome. One is very stable and withstands both temperature and RNase treatment while the second is promptly abolished by both treatments. Data presented here strongly suggest that the RNA domains exposed to the solvent play a fundamental role in the stability of the 3-D structure of the ribosome particle.

Experimental ocular acute hypertension-induced chromatinic alterations in astrocytic cells in rat optic nerve.

The effects of ocular acute hypertension experimentally induced on the astrocyte cells of rat have been studied. Evaluation was made of the damage to the chromatin of those cells by means of cytochemical (haematoxylin-eosin) analysis and of the state of fragmentation of the DNA by means of the TUNEL technique as well as the protective effect of the peroxide scavenger, troxol, on those events.