The pharmacological action of the V( iv ) compound VO(dmpp) 2 was previously evaluated in vivo using obese pre-diabetic Zucker rats treated with this compound for four weeks. Besides the promising ...results regarding biological parameters indicative of insulin-mimetism, a specific biological activity in mitigating impaired lipid metabolism was observed. This work aims at complementing and reinforcing data formerly attained and it represents an attempt to unveil the effect of this compound at the molecular level, particularly on the metabolic profile of different organs. Ex vivo experiments with tissue samples of the brain, liver and skeletal muscle from treated and non-treated obese Zucker rats, using lean Zucker rats as a control, were carried out by using 1 H-HRMAS. The results obtained showed that the lipid liver content, a characteristic of obese rats, significantly decreased after 4 weeks of VO(dmpp) 2 treatment, as demonstrated by the quantification of the respective signals in the 1 H-HRMAS NMR spectra. In the other analyzed tissues, the differences were not statistically significant, but a trend of a decrease of the abnormal metabolite content in treated obese rats was observed. Importantly, the therapeutic dose used showed no renal toxicity. VO(dmpp) 2 was demonstrated to be able to revert the impaired lipid metabolism in vivo and 1 H-HRMAS NMR was revealed to be a good tool to simultaneously assess the effects of a drug on the metabolic profile of different organs and tissues.
Overexpression of transferrin receptors (TfRs), which are responsible for the intracellular uptake of ferric transferrin (Tf), has been described in various cancers. Although molecular biology ...methods allow the identification of different types of receptors in cancer cells, they do not provide features about TfRs internalization, quantification and distribution on cell surface. This information can, however, be accessed by fluorescence techniques. In this work, the quantum dots (QDs)' unique properties were explored to strengthen our understanding of TfRs in cancer cells.
QDs were conjugated to Tf by covalent coupling and QDs-(Tf) bioconjugates were applied to quantify and evaluate the distribution of TfRs in two human glioblastoma cells lines, U87 and DBTRG-05MG, and also in HeLa cells by using flow cytometry and confocal microscopy.
HeLa and DBTRG-05MG cells showed practically the same TfR labeling profile by QDs-(Tf), while U87 cells were less labeled by bioconjugates. Furthermore, inhibition studies demonstrated that QDs-(Tf) were able to label cells with high specificity.
HeLa and DBTRG-05MG cells presented a similar and a higher amount of TfR than U87 cells. Moreover, DBTRG-05MG cells are more efficient in recycling the TfR than the other two cells types.
This is the first study about TfRs in human glioblastoma cells using QDs. This new fluorescent tool can contribute to our understanding of the cancer cell biology and can help in the development of new therapies targeting these receptors.
•We demonstrated an efficient conjugation between QDs and Tf.•We present a specific TfR labeling using nanotechnology in cancer cells.•DBTRG and HeLa cells line present higher amount of TfR when compared to U87.•DBTRG cells are more efficient in recycling the TfR than HeLa or U87 cells.•Our results can help to develop new therapies targeting this receptor.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Intracellular Na+, H+, and ATP and mechanical performance were measured in the isovolumic perfused rat heart during ischemia. The concentration of intracellular sodium, Na+i, was determined by atomic ...absorption spectroscopy under control conditions, and Na+i was monitored by 23Na NMR spectroscopy at 1-min intervals under control conditions and during global ischemia. ATP, H+, and Pi were measured by 31P NMR in a separate group under identical conditions. The control Na+i measured by atomic absorption was 30.7 +/- 3.3 mM (mean +/- SD, n = 6), and Na+i measured by NMR was 6.2 +/- 0.5 mM (n = 3). Brief ischemia (10 min) was associated with a 54% increase in Na+i which reversed completely with reperfusion. Developed pressure also returned to control values upon reperfusion. Prolonged ischemia (30 min) produced continuous further accumulation of sodium (0.53 mM/min, r2 = 0.99). H+ also increased approximately linearly early in ischemia (0.084 microM/min, r2 = 0.97). The rate of increase in Na+i was more than 4000 times greater than the increase in H+ on a molar basis. Nevertheless, H+/Na+i increased early in ischemia because the proportional change in H+ was greater than that in Na+i. These results indicate that (1) intracellular sodium measured by NMR in the functioning heart is about 20% of total intracellular sodium; (2) intracellular acidosis and accumulation of sodium develop simultaneously during global ischemia; (3) increased intracellular sodium content is not in itself an indicator of irreversible injury; and (4) recovery of mechanical performance is associated with return of Na+i (measured by NMR) to baseline after brief ischemia. The mechanism of the increase in sodium content detected by NMR is unknown.
Cell-penetrating peptides (CPPs) have been extensively exploited in gene therapy approaches as vectors for intracellular delivery of bioactive molecules. The ability of CPPs to be internalized into ...cells and their capacity to complex nucleic acids depend on their molecular structure, both primary and secondary, namely regarding hydrophobicity/hydrophilicity. CPP acylation has been used as a strategy to improve this structural feature.
Acyl groups (from 6 to 18 carbon atoms) were attached to the S413-PV peptide and their effects on the peptide competence to complex siRNAs and to mediate gene silencing in glioblastoma (GBM) cells were studied. A systematic characterization of membrane interactions with S413-PV acyl-derivatives was also conducted, using different biophysical techniques (surface pressure-area isotherms in Langmuir monolayers, DSC and 31P NMR) to unravel a relationship between CPP biological activity and CPP effects on membrane stability and lipid organization.
A remarkable concordance was noticed between acylated-S413-PV peptide competence to promote gene silencing in GBM cells and disturbance induced in membrane models, the lauroyl- and myristoyl-S413-PV peptides being the most effective. A cut-off effect was described for the first time regarding the influence of acyl-chain length on CPP bioactivity.
C12-S413-PV showed high capacity to destabilize lipid bilayers, to escape from lysosomal degradation and to mediate gene silencing without promoting cytotoxicity.
Besides unraveling a new CPP with high potential to be employed as a gene delivery vector, this work emphasizes the benefit from allying biophysical and biological studies towards a proper CPP structural refinement for successful pre-clinical/clinical application.
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•The ability of the CPP S413-PV as a siRNA delivery system is enhanced by acylation.•Acyl-S413-PV biological activity shows a bell-shape dependence on acyl chain length.•Lipid membrane disturbance shows a bell-shape dependence on acyl chain length.•C12-S413-PV is the most competent analog in mediating gene silencing in GBM cells.•C12-S413-PV ability may be related to endosomal escape by membrane destabilization.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
For the development of redox responsive MRI probes based on the Mn
III
/Mn
II
couple, stable complexation of both reduced and oxidized forms of the metal ion and appropriate tuning of the redox ...potential in the biologically relevant range are key elements. The water soluble fluorinated Mn-porphyrin derivative Mn-
3
satisfies both requirements. In aqueous solutions, it can reversibly switch between Mn
III
/Mn
II
oxidation states. In the presence of ascorbic acid or β-mercaptoethanol, the Mn
III
form undergoes reduction, which is slowly but fully reversed in the presence of air oxygen. A UV-Vis kinetic study of Mn
III
/Mn
II
reduction under oxygen-free conditions yielded second-order rate constants,
k
2
, of 46.1 M
−1
s
−1
and 13.8 M
−1
s
−1
for the reaction with ascorbic acid and β-mercaptoethanol, respectively. This could correspond, in the absence of oxygen, to a half-life of a few minutes in blood plasma and a few seconds in circulating immune cells where ascorbic acid reaches 20-40 μM and a few mM concentrations, respectively. In contrast to expectations based on the redox potential, reduction with glutathione or cysteine does not occur. It is prevented by the coordination of the glutathione carboxylate group(s) to Mn
III
in the axial position, as was evidenced by NMR data. Therefore, Mn
III
-
3
acts as an ascorbate specific turn-on MRI probe, which in turn can be re-oxidized by oxygen. The relaxivity increase from the oxidized to the reduced form is considerably improved at medium frequencies (up to 80 MHz) with respect to the previously studied Mn-TPPS
4
analogues; at 20 MHz, it amounts to 150%. No
in vitro
cytotoxicity is detectable for Mn-
3
in the typical MRI concentration range. Finally,
19
F NMR resonances of Mn
III
-
3
are relatively sharp which could open further opportunities to exploit such complexes as paramagnetic
19
F NMR probes.
A water-soluble fluorinated Mn
III/II
porphyrin responds reversibly to ascorbate redox state as a turn-on MRI probe.
Type 2 diabetes mellitus has been associated with obesity, metabolic syndrome, cardiovascular diseases and cancer. Attempts have been made for early diagnosis and finding effective drugs to prevent ...severe consequences and ameliorate the symptoms of this disorder. In this work, the pharmacological properties of VO(dmpp)2, bis(1,2-dimethyl-3-hydroxy-4-pyridinonato)oxovanadium(IV), were in vivo evaluated. For 4weeks fatty Zucker rats were subjected to a daily dose of VO(dmpp)2 (44μmol/kg) and their metabolic profile was followed by assessing different biological parameters at established time points: body weight, subcutaneous fat width and hepatic triglyceride content determined by magnetic resonance imaging and spectroscopy, respectively. A glucose tolerance test was performed at the end of the experiment. After treatment, treated obese rats presented a weight significantly lower than the non-treated obese animals (359.0±11.1 vs. 433.5±6.2g, P<0.05), a thinner subcutaneous fat width, and a statistically significant decrease in hepatic triglyceride content (5.41±0.59 vs. 21.03±1.40%, P<0.0005). Additionally, the glucose intolerant profile of fatty Zucker rats was completely reversed in treated animals (102.3±2.1 vs. 172.4±1.3mg/100mL; P<0.0005). These results reinforce the therapeutic action of VO(dmpp)2 which shows particular effects on lipid metabolism.
VO(dmpp)2 reverts different biological parameters indicative of abnormal conditions in a pre-diabetic animal model, like obesity, steatosis and insulin-resistance. Display omitted
► VO(dmpp)2 restored gain of body weight in obese rats to non-pathological values. ► Sub-cutaneous fat width and HTG of obese Zucker rats decreased with VO(dmpp)2. ► VO(dmpp)2 reverts the insulin resistant profile characteristic of obese Zucker rats. ► VO(dmpp)2 demonstrated to be a promising drug for obesity, and insulin resistance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We report a synthetic strategy to combine different moieties in a single structure using cyanuric chloride (2,4,6-trichlorotriazine) as a starting platform for preparing potential bioimaging agents. ...This reacted with macrocycles of the porphyrin family and DOTA type metal chelators through mono-, di- and tri- substitution of its chlorine atoms by appropriate nucleophiles, controlling the stepwise by temperature, to produce a system that opens the potential for biomedicinal applications. Porphyrins were chosen as one of the sensing arms, based on their rich structural chemistry, and excellent photophysical properties, while DO3A was used since it can form a versatile aminopropionate functionalized metal ion chelator. All new compounds were fully characterized, both spectroscopically and photophysically.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
The binding of the V
V
oxidation products of two vanadium(IV) compounds, VO(dmpp)
2
and VO(maltolato)
2
, which have shown promising anti‐diabetic properties, to human serum albumin (HSA) ...in aqueous aerobic solution has been studied by
1
H saturation transfer difference (STD) NMR spectroscopy and computational docking studies. Group epitope mapping and docking simulations indicate a preference of HSA binding to the 1:1 VO
2
(dmpp)(OH)(H
2
O)
–
and 1:2 VO
2
(maltol)
2
–
vanadium(V) species. By using known HSA binders, competition NMR experiments revealed that both complexes preferentially bind to drug site I. Docking simulations carried out with HADDOCK together with restraints derived from the STD results led to three‐dimensional models that are in agreement with the NMR spectroscopic data, providing useful information on molecular interaction modes. These results indicate that the combination of STD NMR and data‐driven docking is a good tool for elucidating the interactions in protein–vanadium compounds and thus for clarifying the mechanism of drug delivery as vanadium compounds have shown potential therapeutic properties.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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