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F Podo

Publications and source records attributed to F Podo.

69 records · Page 4Linked to original sources
PubMed1977

The interaction of histone H3 with histone H4 and with other histones studied by 19F nuclear magnetic resonance.

The behaviour, upon variations in ionic strength, pH and temperature of 19F nuclear nuclear magnetic resonance signals of the trifluoroacetonylated derivative of histone H3 is compared with those of the H3-H4 complex and of the Hv fraction (an equimolar mixture of H2A, H2B, H3 and h4). The line width of the 19F-labelled histone H3 signals increases with ionic strength or pH, an effect consistent with aggregation of the protein. In the case of H3-H4 complex or Hv the line width decreases at intermediate ionic strengths (0.1-0.25 M NaCl). This effect is interpreted as the consequence of the formation of a well defined structure with ionic strength. At high salt concentrations the line width increases as a consequence of the final rigid quaternary structure or of the formation of higher aggregates.

Animals↗
PubMed1977

Nuclear magnetic resonance studies of lecithin bimolecular leaflets with incorporated fluorescent probes.

Pulse Fourier transform proton magnetic resonance techniques have been used to determine (i) the precise location of four membrane fluorescent probe molecules in lipid bilayer model membranes above the gel-to-liquid crystal transition temperature, tm; (ii) the dynamical perturbation of the host lipid molecules induced by incorporation of the probe into the bilayer. The regions of maximal perturbation induced by these probes do not necessarily coincide with their actual location in the bilayer.

Fluorescent Dyes↗
PubMed1976

Proton magnetic relaxation studies of mixed phosphatidylcholine/fatty acid and mixed phosphatidylcholine bimolecular bilayers.

High resolution proton spin-lattice relaxation times (T1), spin-spin relaxation times (T2) and resonance linewidths were measured above the gel-to-liquid crystal transition temperature (Tm), in phosphatidylcholine bilayers possessing various degrees of intramolecular motional anisotrophy at the level of various alkyl chain proton groups. The experiments were designed to test the hypothesis that coupled trans-gauche isomerizations along the chains can be responsible for the anisotropic motion of phosphatidylcholine proton groups in bilayer membranes (Horwitz, A.F., Horsley, W.J. and Klein, M.O. (1972) Proc. Natl. Acad. Sci. U.S. 69,500). Systematic series of structural perturbations of the bilayer were achieved in mixed phosphatidylcholine/fatty acid and in mixed phosphatidylcholine bilayers where the degree of motional anisotrophy of the chains' proton groups was gradually reduced by progressively increasing the chain length disparity of the two components. The systematic T1 and T2 variations observed were interterpreted on the basis of the Woessner's treatment for computing the relaxation times of a spin pair reorienting randomly about an axis which, in turn, tumbles randomly (Woessner, D.E. (1962) J. Chem. Phys. 36, 1). The results confirmed in a qualitative sense the original hypothesis made by Horwitz et al. The time-averaged structural interpretations suggested by the mangetic relaxation studies are in agreement with low-angle X-ray diffraction results obtained below Tm. In addition, the T1 values evaluated at various temperatures in dipalmitoyl phosphatidylcholine vesicles incorporated with either 2H-labeled or unlabeled palmitic acid chains indicated that the average intermolecular contribution to the spin-lattice relaxation rate of the proton groups of the phosphatidylcholine chains appears comparable to the intramolecular term at temperatures moderately higher than Tm, but becomes less and less important as the temperature is further increased above the thermal transition.

Binding Sites↗
PubMed1976

Structural and dynamical studies of mixed chlorophyll/phosphatidylcholine bilayers via x-ray diffraction, absorption polarization spectroscopy and nuclear magnetic resonance.

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidylcholine fatty acids chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a 10(-9)-10(-10)s time scale in the ideally mixed two-component bilayer.

Bacteriochlorophylls↗
PubMed1975

The application of nuclear magnetic resonance spectroscopy to the study of natural and model membranes.

This review article outlines some potentials and limits of the recent application of high resolution Nuclear Magnetic Resonance technique--coupled to the Fourier transformation methods--to the study of biological membranes. Molecular arrangement and dynamical structure characters can be assessed at the level of individual chemical groups in lipid bilayer regions of natural and model membranes, through the determination of physical parameters like chemical shifts, spin-lattice (T1) and spin-spin (T2) nuclear magnetic relaxation times. The results of some significant experiments carried out on single-wall lecithin vesicles as well as on intact natural membranes, are summarized and discussed. Useful information can be obtained on the lipid fatty-acid chains thermal transition, by comparing two lecithin vesicles of the same size, formed by the same host lecithin, but incorporated with different molecular components. In particular, T1 and T2 measurements, interpreted in terms of a two- (or more-) correlation time theoretical models, are able to demonstrate different degrees of motional anisotropy in bilayers formed by mixed lecithins or by mixtures of lecithin and fatty acids, possessing moderately different chain lengths [13]. Chromophore-containing molecules, like chlorophyll [12] or fluorescent probes [14] can be located, within few Angstroms, in a lipid bilayer through proton chemical shift measurements; in addition the perturbation of the lipid membrane structure, as induced by the incorporated probe, is assessed mainly in terms of the intramolecular dynamical structure of the host lecithin molecules, by means of T1 and linewidth studies. The comparison of the n.m.r. relaxation behaviour in intact membranes and in vesicles formed by their extracted lipids may, finally, provide indirect information on the lipid-protein intermolecular interactions and relative mobility, besides indicating the intramolecular mobility characters of the lipid bilayer regions of the membrane.

Fatty Acids↗
PubMedDate not supplied

In vivo 31P MRS of experimental tumours.

More than 50% of cancers fail to respond to any individual treatment and tumour follow-up after treatment plays a major role in routine therapy planning and pharmacological research. Today, MRS is the only technological approach providing non-invasive access to tumour biochemistry. Ten years ago, expectations were raised concerning 31P MRS as an exciting and promising technical approach to the study of tumours. However the expectations have not always come to fruition. How close are we now to seeing routine 31P NMR in clinical oncology? This review of the 127 published papers shows spectroscopy results in more than 150 experimental animal tumour models. These tumour/host/treatment systems provide us with a useful basis to evaluate the current state of the art, summarize the basic knowledge presently available, determine the key points underlying the present disappointment of some clinical oncologists and stimulate new basic research. The information collected concerns the discussion of the reliability of experimental models in oncology, the technical improvement of magnetic resonance technology and the monitoring of bioenergetic status, pH regulation and phospholipid metabolism in treated and untreated tumours. Recent advances (two-thirds of the papers have been published in the last 5 years) seem to provide more optimistic perspectives than those generally accepted a few years ago, in the depressing period following early pioneering work.

Animals↗
PubMedDate not supplied

Identification and characterization of biological tissues by NMR. Concerted research project of the European Economic Community. Introduction, objectives, and activities.

NMR offers, in the complex proton relaxation properties of biological tissues, a unique and potentially powerful tool of tissue characterization. The difficulties encountered so far in comparing the results of in vitro and in vivo NMR studies carried out in different laboratories inhibit NMR methodologies from fully expressing their content of physiopathological information, as well as their potential for therapeutic monitoring. Moreover, obtaining accurate data on proton relaxation in vivo requires accurate location of the anatomical region under study. The acquisition of acceptable information about location and effects is dependent on the establishment of agreed procedures for tests, as well as on the use of appropriate test objects and test substances. A concerted research project entitled "Identification and Characterization of Biological Tissues by NMR" was activated in 1984 by the European Economic Community as part of the Third Medical Research Program, under the auspices of the Biomedical Engineering Concerted Action Committee. A series of papers is presented here which illustrates objectives and scientific programs of this project, the protocols adopted for multi-center comparison of in vitro and in vivo studies, and the results of validating trials, as well as problems of reference substances.

European Union↗
PubMedDate not supplied

Pentose phosphate pathway alterations in multi-drug resistant leukemic T-cells: 31P NMR and enzymatic studies.

31P NMR studies were carried out on the parental drug-sensitive human T-lymphoblastoid cell line CCRI-CEM (CEM) and its multi-drug-resistant (MDR) CEM-VBL100 variants, to assess the role of the pentose phosphate (PP) in MDR expression. CEM and CEM-VBL100 were incubated in the presence of 2-deoxyglucose, as recently proposed by our group (Clin. Chim. Acta 208: 39, 1992). Accumulation of 2-deoxyglucose 6-phosphate was much lower in the drug-resistant than in sensitive cells, indicating PP shunt activation in the MDR variants. This result was confirmed by enzymatic analyses, which demonstrated that, with respect to the parental line, the MDR variant was characterized by a) unaltered hexokinase activity; b) higher glucose 6-phosphate dehydrogenase activity; c) increased levels of reduced glutathione and marked increase of glutathione peroxidase activity after cell exposure to an oxidizing agent (tert-butylhydroperoxide). These results support the view that cell detoxification mechanisms mediated by the pentose phosphate pathway may contribute to the expression of MDR in tumours.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Detection of neutral active phosphatidylcholine-specific phospholipase C in Friend leukemia cells before and after erythroid differentiation.

With respect to normal tissues, 31P NMR spectra of tumors usually exhibit elevated phosphomonoester (PME) and phosphodiester (PDE) signals, arising from phospholipid metabolites such as phosphocholine (PCho) and glycerophosphocholine (GroPCho) (and/or ethanolamine analogues). PME and PDE resonances may undergo significant alterations during tumor growth, at early stages of tumor response to treatment or following cell differentiation and maturation. The enzymatic mechanisms which regulate these alterations are scarcely understood. Recent studies on agonist-induced phosphatidylcholine (PC) hydrolysis by PC-specific phospholipase C (PC-plc) in cells stimulated by hormones or growth factors suggest the hypothesis that repeated transient activations of this enzyme may also contribute to the elevation of PCho levels in tumor NMR spectra. This paper reports the first direct evidence on neutral active PC-plc activity in a tumour cell system, Friend leukemia cells, either in the undifferentiated (FLC) or differentiated state (dFLC). Cell homogenates were incubated in the presence of mixed diheptanoylphosphatidylcholine/sphingomyelin unilamellar vesicles (SLUV), which were previously shown to represent a good substrate for bacterial plc. 31P NMR analyses allowed the simultaneous detection and quantification of phosphorylated metabolites produced in tumor cell homogenates by PC-plc activity, as well by enzymes active in the PC deacylation pathway. With respect to FLC, dFLC homogenates exhibited higher PC-plc activity and lower accumulation of a deacylation product, GroPCho, in agreement with the elevation in the [PCho]/[GroPCho] ratio, already reported in 31P NMR spectra of intact differentiated cells. The direct detection of PC-plc in this cell system opens novel biochemical interpretations on a series of oncological observations, such as a) transient increases in the levels of PCho and PC-derived diacylglycerols reported in immature or in transformed cells in response to agonist-receptor interactions and b) accumulation of mobile lipids in tumor cell membranes and tissues.

Animals↗
PubMedDate not supplied

Magnetic resonance spectroscopy in cancer: phospholipid, neutral lipid and lipoprotein metabolism and function.

A Special Symposium on "Lipid Metabolism and Function in Cancer. Significance of Magnetic Resonance Spectroscopy (MRS) Measurements in Relation to Biochemical Processes and Cellular Control" was held by the EU BIOMED 1 Concerted Action for "Cancer and Brain Disease Characterization and Therapy Assessment by Quantitative MRS", within the 5th International Conference of Anticancer Research (Corfu, 18-20 October 1995). The aims of the Symposium were to discuss present knowledge and problems in the field of phospholipid, neutral lipid and lipoprotein metabolism in cancer, together with the new potential of these inter-linked areas of research arising from advanced in vitro and in vivo MRS methodologies. This guest editorial is intended to introduce the reader of the Proceedings published in this issue, into the stimulating atmosphere of the Symposium, by summarizing some of the exciting proposals, views, ideas and open questions which emerged from presentations and multi-disciplinary, plenary discussions.

Humans↗
PubMedDate not supplied

Detection of phosphatidylcholine-specific phospholipase C in NIH-3T3 fibroblasts and their H-ras transformants: NMR and immunochemical studies.

Although evidence supports constitutive activation of phosphatidylcholine specific phospholipase C (PC-plc) in rastransformed fibroblasts, no studies have been devoted to measure the basal activity levels of this enzyme, its molecular characteristics and subcellular localization. This paper reports for the first time measurements of the activity of different enzymes responsible for PC hydrolysis (PC-plc; phospholipases A2 (pla2) and A1 (pla1)) in homogenates of murine NIH-3T3 fibroblasts (3T3) and their transformants obtained by human H-ras transfection (3T3ras). To this end, 31P NMR analyses were carried out on total cell homogenates, incubated in the presence of mixed diheptanoylphosphatidylcholine: sphingomyelin (DHPC:SM) unilamellar vesicles (SLUV), in which DHPC acts as a suitable substrate for water-soluble lipolytic enzymes. The basal PC-plc activity levels (0.66 +/- 0.14 and 0.38 +/- 0.10 nmol/10(6) cells.hour in 3T3 and 3T3ras fibroblasts, respectively),were substantially higher (over 30-50x) than those reported in the literature for normal mammalian cells (dog heart myocytes). Moreover the PC-plc activity was about 15-30 times lower than the overall PC deacylation activity in both clones. The use of high titer polyclonal antibodies, raised in a rabbit against bacterial PC-plc, allowed identification of one cross-reactive mammalian PC-plc component (M(r) 66 kD) in cell lysates of both 3T3 and 3T3ras fibroblasts, and detection, by indirect immunofluorescence, of its subcellular localization. In control 3T3 fibroblasts (in the late log-phase of growth) the enzyme was exclusively located in the cytosol, while in H-ras transformed cells it was massively exposed on the external side of the membrane. This new finding strongly suggests that the oncogenic product p2Iras is able to induce (or mediate) translocation of PC-plc across the plasma membrane of ras transformed cells, with possible implications not only on cell biochemistry (enhancement of PC-plc activity, and consequent production of intra- and extracellular PCho and accumulation of neutral lipids) but also on cell-cell interaction mechanisms which facilitate tumour invasion and metastasis of oncogene-transformed cells.

3T3 Cells↗
PubMedDate not supplied

Differentiation of glioblastoma multiforme from astrocytomas by in vitro 1H MRS analysis of human brain tumors.

In vitro high resolution 1H NMR spectroscopy allows non-invasive metabolic evaluation of specimens derived from surgically biopsied or resected brain tumors, with the aim of identifying potential markers of different malignancy grading, and improving diagnostic and therapeutic strategies. In the present study we evaluated 36 patients affected by different brain gliomas (7 well differentiated astrocytomas, 7 anaplastic astrocytomas, 16 glioblastomas, 6 oligodendrogliomas). These analyses allowed discrimination between well differentiated and anaplastic astrocytomas (AII + AA) and glioblastoma multiforme (GM) samples on the basis of the ratio between the integrated choline-containing resonance (b"Cho") and the creatine peaks (creatine (Cr) + Phosphocreatine (PCr)). While no definite difference was found between AII and AA, significantly higher values were observed for this ratio in GM. Other signals, derived from different metabolites, such as Glycine (Gly) and N-acetyl-aspartate (NAA), may also assume relevance in differential tumor diagnosis. In this study an increased [Gly]/[Cr + PCr] ratio was observed in GM with respect to AII and AA. The NAA levels observed in our tumor specimens may be explained on the basis of tumor cell infiltration into brain adjacent tissue. Interesting, but inconclusive, are the data concerning oligodendrogliomas, which, also in well differentiated forms, exhibit increased levels of b"Cho"/(Cr + PCr) ratio. The present study confirms the role of MRS in the biochemical characterization of neoplastic brain tissue and its potential contribution to a better selection of multidisciplinary treatment strategies.

Astrocytoma↗