28 research outputs found
Crosstalk between Phosphodiesterase 7 and Glycogen Synthase Kinase-3: Two Relevant Therapeutic Targets for Neurological Disorders
Chronic neuroinflammation has been
increasingly recognized as a primary mechanism underlying acute brain
injury and neurodegenerative diseases. Enhanced expression of diverse
pro-inflammatory agents in glial cells has been shown to contribute
to the cell death that takes place in these disorders. Previous data
from our group have shown that different inhibitors of the cyclic
adenosine monophosphate (cAMP) specific phosphodiesterase 7 (PDE7)
and glycogen synthase kinase-3 (GSK-3) enzymes are potent anti-inflammatory
agents in different models of brain injury. In this study, we investigated
cross-talk between PDE7 and GSK-3, two relevant therapeutic targets
for neurological disorders, using a chemical approach. To this end,
we compared specific inhibitors of GSK-3 and PDE7 with dual inhibitors
of both enzymes with regard to anti-inflammatory effects in primary
cultures of glial cells treated with lipopolysaccharide. Our results
show that the GSK-3 inhibitors act exclusively by inhibition of this
enzyme. By contrast, PDE7 inhibitors exert their effects via inhibition
of PDE7 to increase intracellular cAMP levels but also through indirect
inhibition of GSK-3. Activation of protein kinase A by cAMP results
in phosphorylation of Ser9 of GSK-3 and subsequent inhibition. Our
results indicate that the indirect inhibition of GSK-3 by PDE7 inhibitors
is an important mechanism that should be considered in the future
development of pharmacological treatments
Photoligation of an Amphiphilic Polymer with Mixed Coordination Provides Compact and Reactive Quantum Dots
We introduce a new
set of multicoordinating polymers as ligands
that combine two distinct metal-chelating groups, lipoic acid and
imidazole, for the surface functionalization of QDs. These ligands
combine the benefits of thiol and imidazole coordination to reduce
issues of thiol oxidation and weak binding affinity of imidazole.
The ligand design relies on the introduction of controllable numbers
of lipoic acid and histamine anchors, along with hydrophilic moieties
and reactive functionalities, onto a polyÂ(isobutylene-<i>alt</i>-maleic anhydride) chain via a one-step nucleophilic addition reaction.
We further demonstrate that this design is fully compatible with a
novel and mild photoligation strategy to promote the in situ ligand
exchange and phase transfer of hydrophobic QDs to aqueous media under
borohydride-free conditions. Ligation with these polymers provides
highly fluorescent QDs that exhibit great long-term colloidal stability
over a wide range of conditions, including a broad pH range (3–13),
storage at nanomolar concentration, under ambient conditions, in 100%
growth media, and in the presence of competing agents with strong
reducing property. We further show that incorporating reactive groups
in the ligands permits covalent conjugation of fluorescent dye and
redox-active dopamine to the QDs, producing fluorescent platforms
where emission is controlled/tuned by Förster Resonance Energy
Transfer (FRET) or pH-dependent charge transfer (CT) interactions.
Finally, the polymer-coated QDs have been coupled to cell-penetrating
peptides to facilitate intracellular uptake, while subsequent cytotoxicity
tests show no apparent decrease in cell viability
Effects of VP1.15 and S14 treatment on IκB-α degradation.
<p>By Western Blot analysis, a basal level of IκB-α was detected in the spinal cord from sham-operated animals, whereas IκB-α levels were substantially reduced in SCI mice (a, a1 and b,b1). VP 1.15 and S14 treatment prevented the SCI-induced IκB-α degradation (a, a1 and b,b1 respectively). A representative blot of lysates obtained from each group is shown, and densitometry analysis of all animals is reported. The relative expression of the protein bands from three separated experiments was standardized for densitometry analysis *<i>P</i><0.01 <i>vs.</i> Sham; °<i>P</i><0.01 <i>vs.</i> SCI.</p
Effect of VP1.15 and S14 on TNF-α and IL-1β release and on MPO activity.
<p>A substantial increase in TNF-α (a) and IL-1β (b) production was found in spinal cord tissue collected from SCI mice at 24 h. Spinal cord levels of TNF-α and IL-1β were significantly attenuated by the VP 1.15 and S14 treatment (a, b respectively). Moreover, MPO activity in spinal cord of untreated SCI-operated mice was significantly increased at 24 h after the damage in comparison to sham mice (c). Treatment with VP 1.15 and S14 significantly reduced the SCI-induced increase in MPO activity (c). Data are means ±S.E. mean of 10 mice for each group. *<i>p</i><0.01 vs sham, °<i>p</i><. 0.01 vs SCI.</p
Western blot analysis for Bax.
<p>By Western blot analysis, Bax levels were appreciably increased in the spinal cord from SCI mice (a, a1 and b, b1). On the contrary, VP1.15 (a, a1) and S14 (b, b1) treatment prevented the SCI-induced Bax expression. The relative expression of the protein bands was standardized for densitometric analysis to β-actin levels, and reported in panel a, a1 and b, b1 are expressed as mean ± s.e.m. from n = 5/6 spinal cord for each group. *<i>P</i><0.01 vs sham, °<i>P</i><0.01 vs SCI+vehicle.</p
Purple‑, Blue‑, and Green-Emitting Multishell Alloyed Quantum Dots: Synthesis, Characterization, and Application for Ratiometric Extracellular pH Sensing
We
report the synthesis of a series of Cd<sub><i>x</i></sub>Zn<sub>1–<i>x</i></sub>Se/Cd<sub><i>y</i></sub>Zn<sub>1–<i>y</i></sub>S/ZnS and ZnSe/Cd<sub><i>y</i></sub>Zn<sub>1–<i>y</i></sub>S/ZnS
multishell alloyed luminescent semiconductor quantum dots (QDs) with
fluorescence maxima ranging from 410 to 530 nm which cover the purple,
blue, and green portion of the spectrum. Their subsequent surface
modification to prepare water-soluble blue-emitting QDs, characterization,
and application for ratiometric pH sensing in aqueous buffers and
in an extracellular environment are further described. QDs were synthesized
starting from ZnSe cores, and the fluorescence peak positions were
tuned by (i) cation exchange with cadmium ions and/or (ii) overcoating
with Cd<sub><i>y</i></sub>Zn<sub>1–<i>y</i></sub>S layers. The as-prepared QDs had reasonably high fluorescence
quantum yields (∼30–55%), narrow fluorescence bands
(fwhm ∼25–35 nm), and monodispersed semispherical shapes.
Ligand exchange with hydrophilic compact ligands was successfully
carried out to prepare a series of water-soluble blue-emitting QDs.
QDs coated with the hydrophilic compact ligands preserved the intrinsic
photophysical properties well and showed excellent colloidal stability
in aqueous buffers for over a year. The blue-emitting QDs were further
conjugated with the pH-sensitive dye, fluorescein isothiocyanate (FITC),
to construct a fluorescence resonance energy transfer-based ratiometric
pH sensing platform, and pH monitoring with the QD-FITC conjugates
was successfully demonstrated at pHs ranging between 3 and 7.5. Further
assembly of the QD-FITC conjugates with membrane localization peptides
allowed monitoring of the pH in extracellular environments. High quality,
water-soluble blue-emitting QDs coated with compact ligands can help
expand the practical fluorescence range of QDs for a variety of biological
applications
PDE7 inhibitors and their chemical structures.
<p>a and b represent a chemical structures of VP1,15 and S14respectively. In order in the spinal cord tissue collected from SCI mice, was observed the alteration of morphology of neurons when compared with sham-operated mice (Fig d and c respectively). A protection against alteration of neuron's morphology was observed in mice group treated with VP 1.15 and S14 (e, f respectively).</p
Effects of VP1.15 and S14 on TNF-α and IL-1β expression.
<p>In addition, spinal cord sections were processed at 24 h after SCI to determine the immunohistological staining for TNF-α and IL-1β expression. A substantial increase in TNF-α (b) and IL-1β (f) expression was found in inflammatory cells of the spinal cord tissues from SCI mice at 24<sup>th</sup> hour after SCI. Spinal cord levels of TNF-α and IL-1β expression were significantly attenuated in VP 1.15 (d,h respectively) and S14 (c, g respectively) treated mice in comparison to SCI animals. Densitometry analysis of immunocytochemistry photographs (n = 5 photos from each sample collected from all mice in each experimental group) for TNF-α and IL-1β (<b>i</b>) from spinal cord tissues was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as % of total tissue area. This figure is representative of at least 3 experiments performed on different experimental days. *<i>p</i><0.01 vs. Sham. °<i>p</i><0.01 vs SCI+vehicle. ND: not detectable</p
Effect of PDE7 inhibitors treatment on histological alterations of the spinal cord tissue 24 h after injury.
<p>A significant damage to the spinal cord, from SCI operated mice at the perilesional area, was assessed by the presence of edema as well as alteration of the white matter 24 h after injury (b and b1, see histological score e). Notably, a significant protection from SCI damage was observed in the tissue collected from VP1.15 and S14 treated mice (c, c1 and d, d1 respectively, see histological score e). The histological score (<b>e</b>) was made by an independent observer. wm: White matter; gm: gray matter. This figure is representative of at least 3 experiments performed on different experimental days. Values shown are mean ± s.e. mean of 10 mice for each group. *<i>p</i><0.01 vs. Sham. °<i>p</i><0.01 vs. SCI+vehicle.</p
Effects of PDE7 inhibitors on iNOS expression.
<p>iNOS expression was evaluated by immunohystochemical analysis in the spinal cord section 24 h after SCI. Spinal cord section from sham-operated mice did not stain for iNOS (a), whereas spinal cord section obtained from SCI-operated mice exhibited positive staining for iNOS (b) mainly localized in various inflammatory cells in the grey matter. VP 1.15 and S14 treatment (c, d respectively) reduced the degree for positive staining for iNOS in the spinal cord tissue. Densitometry analysis of immunocytochemistry photographs (n = 5 photos from each sample collected from all mice in each experimental group) for iNOS (e) from spinal cord tissues was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as % of total tissue area. ND: not detectable. This figure is representative of at least 3 experiments performed on different experimental days. *<i>p</i><0.01 vs Sham. °<i>p</i><0.01 vs SCI+vehicle.</p