Concentration-dependent Way Research Articles

PCramoll and rCramoll as New Preventive Agents against the Oxidative Dysfunction Induced by Hydrogen Peroxide.

Oxidative stress plays an important role in the induction of cell death and is associated with various pathologic disorders; therefore, the search for natural products that attenuate the effects produced by oxidant agents is greatly increased. Here, the protective effects of native lectin from Cratylia mollis seeds (pCramoll) and recombinant Cramoll 1 (rCramoll) against H2O2-induced oxidative stress in Vero cells were evaluated. Both lectins significantly attenuated the H2O2-induced cytotoxicity in a concentration-dependent way. The maximum protective effects were 96.85 ± 15.59% (rCramoll) and 59.48 ± 23.44% (pCramoll). The Live/Dead analysis showed a reduction in the percentage of dead cells from 65.04 ± 3.29% (H2O2) to 39.77 ± 2.93% (pCramoll) and 13.90 ± 9.01% (rCramoll). The deleterious effects of H2O2 on cell proliferation were reduced to 10.83% (pCramoll) and 24.17% (rCramoll). Lectins treatment attenuated the excessive superoxide production, the collapse of the mitochondrial membrane potential, and the lysosomal and DNA damage in H2O2-treated cells. In conclusion, our results suggest that pCramoll and rCramoll blocked H2O2-induced cytotoxicity through decreasing reactive oxygen species, restoring the mitochondrial potential, preventing the lysosomal damage and DNA fragmentation, and thus promoting cell survival and proliferation.

A Computational Study of Amyloid β-Protein Assembly in Crowded Environments

Alzheimer's disease is strongly associated with aberrant amyloid β-protein (Aβ) assembly into heterogeneous, metastable oligomeric assemblies with structures that have not been experimentally characterized yet. The 40 and 42 amino acids long Aβ40 and Aβ42 are the two predominant Aβ alloforms in the brain. Whereas Aβ40 and Aβ42 oligomer formation from monomeric state is still inaccessible to fully atomistic explicit-solvent molecular dynamics, Aβ40 and Aβ42 oligomers were structurally characterized using discrete molecular dynamics (DMD) and an intermediate-resolution protein model within the DMD4B-HYDRA implicit solvent force field, and the corresponding oligomer size distributions well matched the available in vitro data. In vivo, however, Aβ coexists with other biomolecules in a rather crowded environment. To understand the effect of crowding on Aβ oligomer formation, we used the DMD4B-HYDRA force field and added to an ensemble of 32 monomeric Aβ40 or Aβ42 peptides inert spherical “crowders” with a diameter of 0.5 nm at various concentrations to examine their effect on Aβ40 and Aβ42 oligomerization pathways. Our results show that crowding shifts oligomer size distributions towards smaller oligomer sizes and increases solubility of both peptides in a concentration-dependent way. The effect is stronger for Aβ42, where crowding abolishes the multimodal character of the oligomer size distribution. Our structural analysis revealed that the stability of larger oligomers is compromised by effective osmotic pressure exerted by the crowders, resulting in an increased rate of assembly breakage. While in vivo crowding agents are not inert as the crowders in our study, we here reveal that crowding-induced osmotic pressure strongly affects protein assembly dynamics, which is of significance to the disease.

Novel FTS-diamine/cinnamic acid hybrids inhibit tumor cell proliferation and migration and promote apoptosis via blocking Ras-related signaling in vitro.

Novel FTS-diamine/cinnamic acid hybrids 7a-f were prepared, and their in vitro biological activities were evaluated. It was found that 7c showed the strongest anti-proliferation activities against cancer cells in vitro and significant growth inhibition of tumor in vivo, and more potential for inhibitory selectivity to tumor cells than intermediate 6 and FTS. Furthermore, the anti-proliferative effect of 7c in Lovo cell lines followed a similar pattern, which included a dose-dependent induction of cell apoptosis via the up-regulation of Bax as well as activated caspase-3 and down-regulation of Bcl-2, and the inhibition of cancer cells migration and invasion in a concentration-dependent way. More importantly, 7c could significantly block Ras-related signaling pathways, which may contribute to its pro-apoptotic induction of the cancer cell lines and its inhibition of carcinoma cell proliferation, migration, and invasion. Therefore, our novel findings may provide a new framework for the discovery of new FTS hybrids for the intervention of human carcinoma cells.

All-Thiol HMGB1 Is a Critical Inducer of Anemia in Sepsis Survivors through CXCR4 Signaling

Introduction: Each year, at least 500,000 patients will survive acute sepsis. Anemia is extremely common in sepsis survivors, further interfering with quality of life and activities of daily living. The pathogenesis of anemia of sepsis is unclear. High mobility group box 1 (HMGB1) is a necessary and sufficient inductor of persistent inflammation in sepsis survivors. After translocation form the nucleus to the cytoplasm, HMGB1 undergoes epigenetic changes in response to environmental queues. Redox state is the best well understood inducer of those modifications. In experimental sepsis, the reduced all-thiol form is the predominant form throughout the first four weeks of sepsis. We recently found that HMGB1 is crucial for the development and maintenance of anemia in sepsis in part by inducing a block in differentiation of late erythropoiesis. Here we present evidence indicating a critical role of the all-thiol HMGB1 in anemia of sepsis survivors through a CXCR4-dependent mechanism.Methods and results: In order to investigate the kinetics of anemia of sepsis survivors, we first induced severe polymicrobial abdominal sepsis by surgical cecal ligation and puncture (CLP) in BALB/c mice. Sham-operated mice were used as controls. Within three days after CLP, hematocrit decreased significantly in CLP survivors three days after CLP (p≤0.001), and hemoglobin (Hb) decreased significantly within 5-7 days. Initially normocytic and normochromic, mean corpuscular hemoglobin (MCH) level was reduced significantly 15 days after CLP, while MCV dropped significantly 20 days after CLP. Sepsis-induced anemia persisted for at least 30 days. Administration of an anti-HMGB1 neutralizing monoclonal antibody (nmAb) reversed anemia. Daily administration of recombinant HMGB1 for one week was sufficient to significantly reduce Hb(p≤0.05), and MCH (p≤0.01).To better understand the role of HMGB1 during erythropoiesis, we isolated CD34+ cells from cord blood and derived them in a methylcellulose-based culture, as well as in three phase liquid culture systems. In methylcellulose, all-thiol HMGB1 (the inflammatory form secreted during necrosis) significantly reduced BFU-E formation (P≤0.0001) in a concentration-dependent way, while sulphonyl-HMGB1 (the fully oxidized form released during apoptosis) had no effect. Moreover, all-thiol HMGB1 disrupted the architecture of BFU-E colonies. In liquid culture, HMGB1 significantly reduced CD34+ cell proliferation (Fig. 1A), and reduced the proportion of GPA+ cells by flow cytometry. In the liquid culture system, all-thiol HMGB1 reduced the proportion of GPA+, CD71+ cells by D16. Recently, CXCR4 was recognized as the receptor needed for all-thiol HMGB1 signaling, after forming a heterodimer with CXCL12. In the three-phase culture system, all-thiol HMGB1 significantly reduced the proportion of GPA+, CD71+ cells, and a similar level of inhibition was found when interfering with an anti-CXCR4 nmAb, CXCR4 by adding CXCL14 (a competitive inhibitor of CXCL12), or a small molecule (AMD3100) (Fig. 1B). In the presence of HMGB1, adding CXCL12 failed to rescue erythropoiesis.Conclusions: Our findings suggest that all-thiol HMGB1, the redox form present during the first weeks after sepsis onset, has a critical role in initiating and sustaining anemia in sepsis survivors. Further, all-thiol HMGB1 interferes with late erythropoiesis through a CXCR4-dependent mechanism. This has potential therapeutic implications for a persistent disorder for which the usual support –including erythropoietin and transfusions- fails to improve outcome, survival, or both. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibitory effects of roscovitine on proliferation and migration of vascular smooth muscle cells in vitro.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are the major cause of in-stent restenosis (ISR). Intervention proliferation and migration of VSMCs is an important strategy for antirestenotic therapy. Roscovitine, a second-generation cyclin-dependent kinase inhibitor, can inhibit cell cycle of multiple cell types. We studied the effects of roscovitine on cell cycle distribution, proliferation and migration of VSMCs in vitro by flow cytometry, BrdU incorporation and wound healing assay, respectively. Our results showed that roscovitine increased the proportion of G0/G1 phase cells after 12 h (69.57±3.65 vs. 92.50±1.68, P=0.000), 24 h (80.87±2.24 vs. 90.25±0.79, P=0.000) and 48 h (88.08±3.86 vs. 88.87±2.43, P=0.427) as compared with control group. Roscovitine inhibited proliferation and migration of VSMCs in a concentration-dependent way. With the increase of concentration, roscovitine showed increased capacity for growth and migration inhibition. Roscovitine (30 μmol/L) led to an almost complete VSMCs growth and migration arrest. Combined with its low toxicity and selective inhibition to ISR-VSMCs, roscovitine may be a potential drug in the treatment of vascular stenosis diseases and particularly useful in the prevention and treatment of ISR.

Impact of graphene oxide on the structure and function of important multiple blood components by a dose‐dependent pattern

Graphene and its derivatives have become great concern in biomedical fields. Though many investigations about their toxicity have been reported, systematic investigation on the interaction with multiple blood components is lacking. In this work, we studied the effects of the graphene oxide (GO) on the structure and function of the blood components, especially, on morphology and hemolysis of red blood cells (RBCs), bovine serum albumin (BSA) and fibrinogen conformation, complement activation, and blood coagulation function. Scanning electron microscopy observation and hemolysis test results showed that the GO can affect RBC morphology and membrane integrity in a concentration-dependent way. Fluorescence and circular dichroism spectra showed that GO could alter the secondary structures and conformation of BSA and fibrinogen. In addition, the presence of GO could also trigger complement activation by detecting their key biomarker molecules in plasma. In the blood clotting process, the GO showed significant adverse effect on the activated partial thromboplastin time but not on prothrombin time of the platelet-poor plasma. Meanwhile, the GO also caused abnormal thromboelastography parameters of the whole blood coagulation. The results obtained in this study provides good insight into understanding the biomedical application of GO in vivo.

Addition of methionine and low cultivation temperatures increase palmitoleic acid production by engineered Saccharomyces cerevisiae.

Palmitoleic acid (POA) has recently gained attention for its health benefits and as a potential resource for industrial feedstock. This study focused on the use of Saccharomyces cerevisiae, which has a high POA content but low lipid content, for POA production. We created an oleaginous S. cerevisiae as a dga1 mutant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np). This was performed to further increase POA content in the oleaginous S. cerevisiae through optimization of culture conditions and genetic modifications. We found that high concentrations of methionine (2.0 g/l) increased POA production in a concentration-dependent way, while other amino acids such as cysteine, glycine, and glutamine showed no effect. It was not clear if the effect of methionine was mediated through S-adenosylmethionine, mainly because its addition did not increase POA content as did the addition of methionine. We increased POA content up to 55% by incubation of the dga1 transformant in a medium containing 2 g/l methionine at lower than normal temperatures ranging from 20 to 25 °C. Cultivation at such temperatures increased dry cell weight, but did not affect the lipid content, thereby increasing total POA production. The effects of methionine and low temperatures (20-25 °C) on POA content were more apparent in the strains overexpressing Dga1∆Np than those harboring empty vectors, which was consistent with the observation that POA was enriched in triacylglycerol. Overexpression of Ole1p, the enzyme responsible for POA production, did not increase POA content of the dga1 mutant overexpressing Dga1∆Np, but increased that of the wild-type strain overexpressing Dga1∆Np. The results suggested that genomic Ole1p in the dga1 mutant was active enough to achieve the optimal POA production under these conditions. Finally, the POA production by the S. cerevisiae transformant was increased 2.5-fold, which demonstrates that oleaginous S. cerevisiae is a potential source of POA.

Pharmacokinetic interaction between nevirapine and nortriptyline in rats: inhibition of nevirapine metabolism by nortriptyline.

One of the most frequent comorbidities of HIV infection is depression, with a lifetime prevalence of 22 to 45%. Therefore, it was decided to study a potential pharmacokinetic interaction between the nonnucleoside reverse transcriptase inhibitor nevirapine (NVP) and the tricyclic antidepressant nortriptyline (NT). NVP and NT were administered to rats either orally, intraduodenally, or intravenously, and the changes in plasma levels and pharmacokinetic parameters were analyzed. Experiments with rat and human hepatic microsomes were carried out to evaluate the inhibitory effects of NT on NVP metabolism. NVP plasma concentrations were significantly higher when this drug was coadministered with NT. The maximum plasma concentrations of NVP were increased 2 to 5 times and the total plasma clearance was decreased 7-fold in the presence of NT. However, statistically significant differences in the pharmacokinetic parameters of NT in the absence and presence of NVP were not found. In vitro studies with rat and human hepatic microsomes confirmed the inhibition of NVP hepatic metabolism by NT in a concentration-dependent way, with the inhibition being more intense in the case of rat microsomes. In conclusion, a pharmacokinetic interaction between NVP and NT was detected. This interaction was a consequence of the inhibition of hepatic metabolism of NVP by NT. In vivo human studies are required to evaluate the effects of this interaction on the pharmacokinetics of NVP before it can be taken into account for patients receiving NVP.

Evaluation of the intestinal permeability and cytotoxic effects of cylindrospermopsin

Cylindrospermopsin is a freshwater and widespread cyanotoxin considered hazardous for human health. Climate change and eutrophication are the main factors influencing the increasing presence of cylindrospermopsin producers that can contaminate human and animal drinking waters, leading to a rise in ecological and human risk. In order to reach the bloodstream and thus the target receptor, an orally administered drug must first cross the intestinal barrier. The goal of this study was to examine the cylindrospermopsin intestinal permeability and its cellular effects on intestinal and hepatic cells. We explored the human intestinal permeability of cylindrospermopsin by performing in vitro permeation studies across the Caco-2 cell monolayer. Cell permeability data indicated a limited passage of the toxin through the intact intestinal epithelium in a time and concentration dependent way. Cylindrospermopsin induced neither damage on the integrity of the monolayer nor cytotoxicity in tests performed with Caco-2 even at micromolar concentration. Opposite, when hepatic Clone 9 cells were exposed to cylindrospermopsin, a noticeable cytotoxicity was observed being more marked at the higher concentrations used. In addition, this cell line showed alterations in reduced glutathione content due to cylindrospermopsin over time. Meanwhile glutamate cysteine ligase levels, the first rate-limiting enzyme of the glutathione route, showed a significant increase. Therefore our results indicate that cylindrospermopsin cytotoxicity is unrelated to protein inhibition or a decrease of reduced glutathione levels in Clone 9 cells.

Inhibitory effect of pumpkin protein on expression of Notch signal in RPMI8226 myeloma cells

This study was aimed to explore the inhibitory effect of pumpkin protein (cucurmosin, CUS) on proliferation of RPMI8226 myeloma cells in vitro and its mechanism. Western blot was used to detect the expression level of Notch-1, Jagged-2, P-Akt and NF-KB in the myeloma cells treated by different concentrations of CUS. The results demonstrated that CUS could down-regulate the protein expression levels of Notch1, Jagged-2, P-Akt and NF-KB in the myeloma cells and with time-and concentration-dependent way, at the same time CUS could also decrease the expressions of BCL-2 and P-Akt. It is concluded that CUS can obviously inhibit the RPMI8226 cell proliferation in vitro, down-regulate the expression levels of Notch signal and its down-stream target genes. Therefore, Notch signaling pathway can be used as a new treatment target for multiple myeloma, and CUS may be become a potential new drug for regulating Notch signaling pathway.

Interleukin-6 Contributes to the Paracrine Effects of Cardiospheres Cultured from Human, Murine and Rat Hearts

Cardiosphere-derived cells (CDCs) were cultured from human, murine, and rat hearts. Diluted supernatant (conditioned-medium) of the cultures improved the contractile behavior of isolated rat cardiomyocytes (CMCs). This effect is mediated by the paracrine release of cytokines. The present study tested the hypothesis, that the cardiovascular state of the donor's heart influences this effect on CMCs and tries to identify the responsible factors. CDCs were cultured from human tissue samples of cardiac surgery and from murine and rat hearts. The supernatants of cultured CDCs from hypertensive humans and rats showed a higher improvement of the contractile behavior of CMCs compared to CDCs of normotensive origin. Subsequently, the cytokine profile of the supernatants was analyzed. Among the cytokines elevated in supernatants originating from hypertensive humans or rats was Interleukin-6. CDCs were also generated from Interleukin-6(-/-) -mice and their wildtype littermates. The supernatant of the cultured Interleukin-6(-/-) -CDCs had no effect on the contractile behavior, whereas the supernatant of the Interleukin-6(+/+) -CDCs showed a positive effect. To confirm the hypothesis that Interleukin-6 contributes to the paracrine effects, CMCs were incubated with Interleukin-6. It improved the contractile function in a concentration dependent way. Finally, the effect of the supernatant of cultured CDCs derived from a hypertensive human sample could be abolished by simultaneous incubation with a specific Interleukin-6 antibody. CDCs release cytokines that improve the contractile behavior of CMCs. This effect is more intense in CDCs from hypertensive donors. Interleukin-6 is involved in this phenomenon.

Rapid and highly-sensitive melamine sensing based on the efficient inner filter effect of Ag nanoparticles on the fluorescence of eco-friendly ZnSe quantum dots

A very practical and competitive sensing strategy for melamine has been developed based on the efficient inner filter effect (IFE) of Ag nanoparticles (AgNPs) on the fluorescence of eco-friendly ZnSe quantum dots (QDs). The fluorescence emission of ZnSe QDs could be quenched significantly by AgNPs via IFE, which resulted in a fluorescence “off-state”. The presence of melamine could induce the aggregation of AgNPs and modulate the IFE process. Thus the IFE-decreased fluorescence could gradually recover in a melamine concentration-dependent way, thereby achieving the fluorescence “turn-on” sensing for melamine. The detection mechanism was clearly illustrated and the influences of various experimental conditions were also investigated. Under the optimal conditions, a decent linear relationship for melamine was obtained in the range from 1.0 to 36μgL−1, and the detection limit was as low as 0.11μgL−1. The IFE-based strategy was successfully applied for melamine analysis in raw milk and egg samples and satisfactory results have been achieved. Therefore, it is very promising to use this simple, rapid, sensitive and eco-friendly method for on-site and real-time screening of melamine.

Role of BCL-2, caspase-3 and NF-κB in astragaloside inducing apoptosis of human NB4 cells

This study was purposed to investigate the apoptosis-inducing effect of astragalosides on acute promyelocytic leukemia(APL) cell line NB4 and its mechanism. NB4 cells were treated with different concentrations (200, 300, 400 µg /ml) of astragalosides for 48 h. The cell proliferation was assayed by using CCK-8 method; the cell apoptosis was analyzed by flow cytometry with Annexin V-FITE/PI double staining. The mRNA expression of BCL-2 and the relative activity of BCL-2, NF-κB and caspase-3 were detected by RT-PCR and Western blot, respectively. The results showed that after treated with astragalosides for 48 h, astragalosides inhibited NB4 cell proliferation in concentration-dependent way, the apoptosis rate of NB4 cells gradually elevated from 4.69% to 40.85% with the increasing of astragalosides concentration. Simultaneously, the mRNA expression of BCL-2 was down-regulated, Western blot analysis showed that the protein expression levels of BCL-2 and NF-κB decreased after astragalosides treatment, while caspase-3 protein expression level increased. It is concluded that the molecular mechanism of the astragalosides-induced apoptosis in NB4 cells may be associated with down-regulation of the expression of BCL-2 and NF-κB, finally the relative activity of caspase-3 activated.

Ellagic Acid: An Unusually Versatile Protector against Oxidative Stress

Several aspects related to the antioxidant activity of ellagic acid were investigated using the density functional theory. It was found that this compound is unusually versatile for protecting against the toxic effects caused by oxidative stress. Ellagic acid, in aqueous solution at physiological pH, is able of deactivating a wide variety of free radicals, which is a desirable capability since in biological systems, these species are diverse. Under such conditions, the ellagic acid anion is proposed as the key species for its protective effects. It is predicted to be efficiently and continuously regenerated after scavenging two free radicals per cycle. This is an advantageous and unusual behavior that contributes to increase its antioxidant activity at low concentrations. In addition, the ellagic acid metabolites are also capable of efficiently scavenging a wide variety of free radicals. Accordingly, it is proposed that the ellagic acid efficiency for that purpose is not reduced after being metabolized. On the contrary, it provides continuous protection against oxidative stress through a free radical scavenging cascade. This is an uncommon and beneficial behavior, which makes ellagic acid particularly valuable to that purpose. After deprotonation, ellagic acid is also capable of chelating copper, in a concentration dependent way, decreasing the free radical production. In summary, ellagic acid is proposed to be an efficient multiple-function protector against oxidative stress.

The orally active male contraceptive agent H2‐gamendazole interacts with organic anion transporting polypeptides expressed in human hepatocytes (1064.18)

The non‐hormonal male contraceptive H2‐gamendazole (H2‐GMZ) is a lonidamine derivative that has been shown to accumulate in rat testes at levels about 10 times higher than in the liver and other organs. Given that H2‐GMZ is an organic anion with a molecular weight of more than 400 Da, it could interact with Organic Anion Transporting Polypeptides (OATPs). Therefore, we wanted to test to what extent H2‐GMZ could interact with OATP1B1 and OATP1B3, two OATPs expressed in human hepatocytes. Uptake of the model substrates estradiol‐17β‐glucuronide, estrone‐3‐sulfate, taurocholate, and of pravastatin was measured in Chinese Hamster Ovary (CHO) cells expressing OATP1B1 or OATP1B3 in the absence and presence of H2‐GMZ. Furthermore, direct uptake of the fluorescent FITC‐H2‐GMZ and of the radiolabeled 3H‐H2‐GMZ was determined in wild‐type and OATP‐expressing CHO cells. Uptake of all OATP substrates was inhibited by H2‐GMZ in a concentration dependent way. Uptake of FITC‐H2‐GMZ was highest in OATP1B3‐expressing CHO cells followed by OATP1B1‐expressing CHO cells. However, when 3H‐H2‐GMZ was used as a substrate, no OATP‐mediated uptake could be detected which is consistent with the in vivo animal data that liver uptake is minimal. Given that fluorescein is a substrate for both OATPs, we assume that in the case of FITC‐H2‐GMZ the fluorescent moiety is recognized by the transporters. Thus, although H2‐GMZ inhibits OATP‐mediated uptake of several substrates in a concentration dependent way, it does not seem to be a good substrate for OATP1B1 and OATP1B3 and liver accumulation in humans is unlikely.

Molecular mechanisms of leptin and pro-apoptotic signals induced by menadione in HepG2 cells

Apoptosis is a significant physiological function in the cell. P53 is known as tumor suppressor cellular factor, executive caspases are also the most involved pathway for apoptosis. Menadione (VK3) has apoptotic action on many harmful cells, but the molecular role of adipokines is not studied enough in this regard, so the ability of menadione to modify the adipokine (leptin hormone), caspase-3 and P53 signals to induce its apoptotic action on HepG2 cells was studied. The study revealed that menadione has anti-viability and apoptotic effect at sub-G1 phase of HepG2 cell cycle. Its cytotoxic effect is mediated by molecular mechanisms included: inhibiting leptin expression and level, activating caspase-3 pathway and up-regulating the expression of P53. Menadione exerts its apoptotic mechanisms in a concentration and time dependent way through ROS generation. In addition to the known apoptotic pathways, the results indicate that suppressing leptin pathway is a significant mechanism for menadione apoptotic effect which made it as a potential therapeutic vitamin in preventing hepatocyte survival and proliferation.

Gold nanoparticle modifies nitric oxide release and vasodilation in rat aorta

Nitric oxide (NO) plays an important role on several biological functions. Recently, it has been reported the possibility of modifying the NO release profile from the NO donors through its coupling to gold nanoparticles (AuNPs). Thus, AuNPs were synthesized and they were exposed to the NO donor ruthenium complex Cis-[Ru(bpy)2(NO)(4PySH)].(PF6)3 termed (Ru-4PySH)-forming AuNPs-{Ru-4PySH}n cluster. Our results indicate that AuNPs do not modify the maximum effect (ME) and potency (pD2) in the vasodilation induced by Ru-4PySH. Both complexes induce similar vascular relaxation in concentration-dependent way. However, the NO released from the complex AuNPs-{Ru-4PySH}n is lower than Ru-4PySH. Both complexes release only NO(0) specie, but AuNPs-{Ru-4PySH}n releases NO in constant way and exclusively in the extracellular medium. In time-course, Ru-4Py-SH was faster than AuNPs-{Ru-4PySH}n in inducing the maximum vasodilation. Inhibition of soluble guanylyl cyclase (sGC) abolished the vasodilation induced by Ru-4PYSH, but not by AuNPs-{Ru-4PySH}n. Non-selective potassium (K(+)) channel blocker TEA had no effect on the vasodilation induced by AuNPs-{Ru-4PySH}n, but it reduced the potency to Ru-4PySH. In conclusion, our results suggest that AuNPs can reduce the permeability of NO donor Ru-4PySH due to AuNPs-{Ru-4PySH}n cluster formation. AuNPs reduce NO release, but they do not impair the vasodilator effect induced by the NO donor. Ru-4PySH induces vasodilation by sGC and K(+) channels activation, while AuNPs-{Ru-4PySH}n activates mainly sGC. Taken together, these findings represent a new pharmacological strategy to control the NO release which could activate selective biological targets.

Impact of homocysteine-thiolactone on plasma fibrin networks

Epidemiologic studies have shown that hyperhomocysteinemia is an independent risk factor for vascular disease. Homocysteine (Hcy) circulates as different species, mostly protein bound, and approximately 1% as its reduced form and the cyclic thioester homocysteine-thiolactone (HTL). Despite the level of plasma thiolactone being markedly low, detrimental effects are related to its high reactivity. HTL reacts with proteins by acylation of free basic amino groups; in particular, the epsilon-amino group of lysine residues forms adducts and induces structural and functional changes in plasma proteins. In order to assess the effects of HTL on plasma fibrin networks, a pool of normal plasma incubated with HTL (100, 500 and 1,000 μmol/L, respectively) was evaluated by global coagulation tests and fibrin formation kinetic assays, and the resulting fibrin was observed by scanning electron microscopy. HTL significantly prolonged global coagulation tests in a concentration-dependent manner with respect to control, and increases were up to 14.5%. Fibrin formation kinetic parameters displayed statistically significant differences between HTL-treated plasma and control in a concentration-dependent way, showing higher lag phase and lower maximum reaction velocity and final network optical density. Electron microscopy analysis of HTL plasma networks revealed a compact architecture, with more branches and shorter fibers than control. We can conclude that HTL induced a slower coagulation process, rendering more tightly packed fibrin clots. Since these features of the networks have been related to impaired fibrinolysis, the N-homocysteinylation reactions would be involved in the prothrombotic effects associated to hyperhomocysteinemia.

Non-monotonic concentration–response relationship of TiO2 nanoparticles in freshwater cladocerans under environmentally relevant UV-A light

The effects of UV-A on the toxicity of TiO2 nano-particles (NPs) were evaluated using Moina macrocopa and Daphnia magna under environmentally relevant level of UV-A. The waterfleas were exposed to TiO2 NPs with different sizes of ~298nm, ~132nm, or ~72nm for up to 48h, with or without UV-A light. Whole body reactive oxygen species and transcription of antioxidant enzyme genes were measured, as well as the survival of the waterflea. In the presence of UV-A, the survival rates of M. macrocopa significantly decreased in concentration dependent way until ~1mg/L TiO2 NPs, but the survivals were reversed at greater concentrations. This peculiar non-monotonic trend of concentration–response relationship might be explained by changes of particle size under different light conditions. TiO2 NPs within a certain size range could be trapped in the filter apparatus and exert toxicity, and the NPs of greater size were subject to either precipitation or ingestion leading to no or little toxicity. Observed TiO2 toxicity was associated with oxidative stress in the filter apparatus. The results of this study showed that the size change due to UV-A irradiation should be considered in evaluation of ecological risks of TiO2 NP.

Comparison of effective α-tocopherol concentrations ameliorating reduced glucose transport and counteracting t-butylhydroxyperoxide-induced lipid peroxidation in isolated erythrocytes from ß-thalassemic patients

In thalassemia patients Red Blood Cell (RBC) membrane is the major target of oxidative stress caused by hemichrome radicals. Moreover, RBC membranes from thalassemic patients are more susceptible to oxidative stress, which causes lipid peroxidation, membrane rigidity, and dysfunction of membrane proteins. We investigated hemoglobin-free isolated RBC membranes (ghosts) from 28 β-thalassemia major patients and from 24 normal blood samples. Our research aimed to determine the effective concentrations of racemic α-tocopherol i) counteracting experimental oxidative stress induced by 2mM tertiary butylhydroperoxide (t-BHP) and ii) ameliorating impaired glucose transport in RBC ghosts from thalassemia patients. Reduced glucose transport in thalassemic ghosts was ameliorated significantly by tocopherol with a maximum effect at 75 ppm concentration. Furthermore, ghosts were pre-incubated with/without α-tocopherol up to 200 ppm before treated with t-BHP as the oxidative agent and reactive membrane thiols determined. As a parameter of lipid peroxidation (LPO) thiobarbiturate-reactive substances were measured and expressed as malondialdehyde levels. Tocopherol counteracted LPO continuously in a concentration-dependent way up 200 ppm. Hence, it is concluded that tocopherol acts at different concentrations as an antioxidant and on glucose transport by GLUT1 in RBC membranes.