1) Study of new biosensors for
intracellular cations, in particular for magnesium and zinc:
characterization of their spectroscopic features and of their
biological application, with particular interest to the magnesium
cellular and tissutal homeostasis, in physiological and
pathological conditions. This study is based on the integration of
different techniques, because it considers the feasibility of
spectroscopic, microscopic and cytofluorimetric approaches for the
assessment of cell magnesium and its fluxes, comparing the results
of magnesium analysis in blood cells of healthy donor and of
patients of selected diseases with 31P Ã¢â‚¬â€œNMRS assays
Ã¢â‚¬Å“in vivoÃ¢â‚¬. Characterization of new fluorescent probes for
Cadmium, to employ in toxicological assays.
2) Study of application of X-ray
microscopy to intracellular ion mapping.
3) Study of cellular magnesium homeostasis and of the role
played by this cation in several aspect of cellular biology, mainly
in cell cycle progression od normal and transformed cells,
apoptosis and Multi-Drug Resistance.
4) Study of antiproliferative e MDR revertant effects of
potential anticancer drugs.
The research on new fluorescent biosensors for
intracellular cations, mainly for magnesium, has been focussed on a
class of fluorescent dyes based on diaza-18-crown-6 appended with
two hydroxyquinoline groups, bearing different atoms in position 5.
In particular the two molecules bearing H (DCHQ1) o Cl (DCHQ2)
substituents showed high affinity and specificity for magnesium and
no interference with calcium, as displayed by commercial probes
such as Mag-Fura and Mag-fluo. The characterization of the DCHQ1 is
quite advanced: DCHQ1 is well tolerated by cells, allows to
evaluate intracellular total magnesium and monitor fast
intracellular magnesium transients after mitochondrial stimuli.
Therefore, this molecule has the potential to be successfully
employed in the study of magnesium homeostasis, representing in
addition, an alternative to atomic absorption spectroscopy (AAS) in
total intracellular magnesium evaluation, especially when
biological specimens of reduced dimensions are available.
The photochemical characterization of DCHQ2 is quite completed
and now we are studing a new derivatives; among them a
DCHQ-AM, which could be entrapped within the cells.
The biochemical and photochemical characterization of DCHQ
probes and their derivatives are carried out to assay their
applicability to total intracellular magnesium and its fluxes by
means of spectrofluorimetry, fluorescence microscopy and flow
cytometry. Thwese studies are carried on in collaboration with
Prof.F.I. Wolf of the UniversitÃ Cattolica di Roma and
with Prof. J. Meier of the UniversitÃ Statale di
The next research projects will focus on: i) application
of the dye DCHQ1 to the evaluation of the total intracellular
magnesium to blood cells, comparing the measures obtained with this
dye in erythrocytes, platelets and lymphocytes of healthy donors
with those obtained by AAS. Similar determinations will be
performed with the other members of the DCHQ family that have
showed suitable characteristics similar to DCHQ1; ii)
application of these assays on patients affected by pathologies
where a deficit in plasma magnesium levels has been reported.
However, it is not yet clarified how this alteration influences the
intracellular levels of total and free intracellular magnesium in
different tissues. The tissue distribution of magnesium will be
performed by different spectroscopic techniques, as 31P-MRS and
standard techniques (AAS, colorimetry and Mag-Fura
fluorimetry). In particular, fluorescence spectroscopy and
flow cytometry will be used to evaluate magnesium by commercial
probes (Mag-fura and/or Mag-fluo) and by DCHQ probes in blood cells
(lymphocytes, erythrocytes and platelets), and the results will be
integrated by free cytosolic Mg2+ measured in brain and in
skeletal muscle by 31P-MRS by Prof. S. Iotti of Dipartimento di
Medicina Interna, dell'Invecchiamento e Malattie
Nefrologiche, University ofi Bologna; iii) study of
other derivatives of Hydroquinoline specific for other
cations of biological interest, as Zn; iv) study of the
applications of X-ray microscopy toimage the intracellular
topography and the valence status of a variety of atomic elements
with high spatial resolution, in collaboration with Prof. S.
Lagormasino of CNR, Rome.