P. Grimaldi

Structural changes induced in proteins by therapeutic ultrasounds

The structural effect induced by therapeutic ultrasound on proteins in aqueous solution has been investigated with FTIR spectroscopy, UV-VIS spectroscopy, circular dichroism and light scattering. Six proteins (cytochrome, lysozyme, myoglobin, bovine serum albumin, trypsinogen, and alpha-chymotrypsinogen A) with different molecular weight and secondary structure have been studied. The experiment has been performed using an ultrasound source at resonant frequency of 1 MHz and sonication times of 10, 20, 30, 40, 50, and 60 min. A different behaviour of proteins under sonication depends on the dominant secondary structure type (alpha-helix or beta-sheets) and on the grade of the ordered structure. The results suggest that the free radicals, produced by water sonolysis, have an important role in the changes of structural order.

UVB-Radiation-Induced Apoptosis in Jurkat Cells: A Coordinated Fourier Transform Infrared Spectroscopy-Flow Cytometry Study

Abstract Pozzi, D., Grimaldi, P., Gaudenzi, S., Di Giambattista, L., Silvestri, I., Morrone, S. and Congiu Castellano, A. UVB-Radiation-Induced Apoptosis in Jurkat Cells: A Coordinated Fourier Transform Infrared Spectroscopy-Flow Cytometry Study. Radiat. Res. 168, 698–705 (2007). We studied the induction of apoptosis in Jurkat cells by UVB radiation (wavelength 290–320 nm) at a dose of 310 mJ/ cm2. We combined Fourier transform infrared (FTIR) spectroscopy with flow cytometry to determine whether the combination of both techniques could provide new and improved information about cell modifications. To do this, we looked for correspondences and correlations between spectroscopy and flow cytometry data and found three highly probable spectroscopic markers of apoptosis. The behavior of the wave number shift of both the Amide I β-sheet component and the area of the 1083 cm−1 band reproduced, with a high correlation, the behavior of the early apoptotic cell population, while the behavior of the Amide I area showed a high correlation with the early plus late apoptotic cell population.

Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: a contribution to the optimization of gene and drug delivery.

Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

The Ultrasound effects on non tumoral cell line at 1 MHz therapeutic frequency

The aim of this research is to investigate some bioeffects due to Therapeutic Ultrasound (1 MHz and 50<IPA<60 W/cm2) which could allow to enhance drugs or genes delivery in non tumoral cells. Ultrasound (US) has been demonstrated to alter the cell membrane permeability due to a biophysical mechanism, Sonoporation, and exploited as a promising non-invasive gene transfer method. We have used the NIH-3T3 cell line as a model system and exposed it to US medical equipment for 15, 30, 45, 60 minutes at distances of 10 and 15 cm from the source transducer, corresponding to the far field region where cm. We have worked with the maximum power in pulsed system with 75% duty cycle. Characterization of the unfocused, planar and with a circular geometry 1 MHz source transducer, was performed and the acoustics pressure was measured by a calibrated 0.5 mm needle hydrophone; moreover, the pressure field generated by the source transducer was simulated. The US effects on cells were assessed by Fourier transform infrared (FTIR) Imaging with focal plane array (FPA) detector. By the IR analysis, the US exposure on non tumoral cells has induced a change of the intensity for CH2 asymmetric stretching (2924 cm−1) band in the lipid region (3000-2800 cm−1) that it could detect an energy-dependent process. It has already shown that cells invest energy to catalyze lipid movement in order to maintain a specific transmembrane phospholipid distribution. Although asymmetry is the rule for control cells, the loss of asymmetry could be associated with the permeability change of plasma membrane inducing temporary pores.

L’accompagnement par les plantes dans le voyage entre les vies

Chaque jour, dans le monde, quelques 200 000 personnes décèdent. Certaines meurent d’accident, d’autres sont tuées. D’autres meurent d’avoir trop consommé, tandis que d’autres meurent de faim. Quelques-unes meurent encore de soif, d’autres noyées, tandis que certaines meurent in utero, d’autres de vieillesse... Chacune d’entre elle est allée à la rencontre de sa propre mort comme elle le pouvait. Quelques-unes ont capitulé et accepté leur destin avec un esprit ouvert et le cœur en paix. D’autres sont mortes dans la confusion, victimes d’une vie non vécue, d’une mort qu’elles ne sont pas parvenues à accepter. Thomas Lewis écrit : « Les pages des avis des décès nous informent que nous mourrons, tandis que les avis des naissances, imprimés en caractères plus petits et à la marge de la page, nous disent que nous serons remplacés, mais de tout cela nous ne saisissons pas la véritable portée. » Nous sommes plus de six milliards d’être bumains, et sommes tous destinés à mourir individuellement. Cette mortalité phénoménale qui concerne chaque année plus de 60 millions d’entre nous, se déroule en quasi secret. Dans moins d’un demi-siècle, ceux qui nous remplaceront auront plus que doublé ces chiffres. Comment fait-on pour garder le secret de tous ces gens qui disparaissent ? Nous devrons renoncer à l’idée que la mort est une catastrophe, un événement détestable ou même étrange, qu’il faut éviter. Tout ce qui vient à la vie semble remplacer quelque chose qui meurt, cellule par cellule. Le problème de la mort, de la douleur et de la maladie restent parmi les plus mystérieux pour l’humanité et, pour l’homme occidental qui a mis de côté la dimension transcendante, le plus angoissant. Même la douleur est niée, ou si peu prise en considération. Et pourtant, en tant que médecins, en tant qu’hommes, nous croisons des yeux implorants, enflammés par la fièvre, éteints par la fatigue, des regards qui interrogent et qui cherchent en vain la raison de la souffrance humaine et qui demandent anxieusement quand et d’où viendra le soulagement.

UV-B radiation induced effects on human T-lymphocytes

The reactive oxygen species (ROS) produced inside cells by UV-B radiation may induce apoptosis, a process that realizes the programmed death of cells. In the present paper the UV induced damage was studied in a human T-Lymphocyte cell line (the Jurkat line) by mean of IR laser photoacoustic spectroscopy (PAS) and by Fourier Transform Infrared Spectroscopy (FTIR) combined with biological assays based on flow cytometry. The apoptosis was induced in vitro in the Jurkat samples by exposition to UV-B radiation with a dose of 310 mJ/cm2. PAS measurements were performed through a 10W c.w. CO2 laser based optical system realised at ENEA Molecular Spectroscopy Laboratory in Frascati (Italy).