mM Of KCl Research Articles

Strategies to Stabilize Dalbavancin in Aqueous Solutions; Section-1: the Effects of Metal Ions and Buffers.

The effect of monovalent (Na+ and K+) and divalent (Ca2+, Mg2+, and Zn2+) metal ions combined with citrate or acetate buffers (pH 4.5) on the stability of dalbavancin in aqueous solutions was investigated. RP-HPLC and HP-SEC were used to evaluate the stability of aqueous solutions of dalbavancin in different combinations of buffers and metal ions after four weeks of storage at 5°C and 55°C. A long-term study of formulations with divalent metal ions was conducted over six months at 5°C., 25°C and 40°C using RP-HPLC. All formulations in citrate buffered solutions precipitated. Dalbavancin solutions in 10mM acetate buffer at 55°C were more stable in 10mM CaCl2, 5mM ZnCl2 and 10mM MgCl2 than those containing 2mM NaCl or 5mM KCl, although the MgCl2 formulations precipitated slightly. No significant effect was observed for any of the divalent metal ions at 40°C for six months. Dalbavancin's stability in solution was improved by a combination of acetate and divalent metal ions at 55°C for four weeks. No effect was observed with acetate or metal ions alone, and no effect was observed after six months at 40°C suggesting that acetate and divalent metal ions together interact with dalbavancin via a thermally activated step to inhibit hydrolysis of the drug.

Extracellular β-d-fructofuranosidase from a novel Aspergillus sp. DHE1 with high potential for biotechnological applications: Purification and biochemical characterization

The current work focused on the purification and biochemical characterization of an extracellular β-d-fructofuranosidase from a novel fungus identified genetically using 18 S rRNA molecular sequencing, as Aspergillus sp. DHE1. The fungus was cultured under solid state fermentation using the Cotton (Gossypium herbaceum) seed waste as substrate. β-d-fructofuranosidase was purified through fractionation by ammonium sulphate salt, ion-exchange chromatography on DEAE-cellulose, and Sephadex G-100 gel filtration, yielding a 13.3-purification fold, 22.6% recovery and 192.9 U/mg protein specific activity. The apparent molecular mass of Aspergillus sp. DHE1 β-d-fructofuranosidase was 78 kDa as estimated on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Optimal pH and temperature were observed at 6.0 and 50 °C, respectively, and the enzyme was quite thermostable at neutral/alkalophilic conditions, retaining 72% of its original activity after 1 h of incubation at 60 °C. The activity of the purified Aspergillus sp. DHE1 β-d-fructofuranosidase was stimulated by 1 mM of KCl (138.5%), NaCl (125.9%), CoCl2 (121.2%), and CaCl2 (112.3%), whereas the same concentration of AgCl, FeCl3, HgCl2, and CuCl2 inhibited the activity by 48.7%, 30.5%, 29.9%, and 26.2%, respectively. The kinetic studies using d-sucrose as the substrate were investigated using a Lineweaver-Burk plot, with Km and Vmax of 0.85 mM and 47.62 U/mL, respectively, indicating the high substrate affinity of β-d-fructofuranosidase. The ability of Aspergillus sp. DHE1 β-d-fructofuranosidase to tolerate ethanol suggests that it may have numerous uses in the food industry, including the production of alcoholic beverages.

G-Quadruplex Aptamer-Ligand Characterization.

In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The KD values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.

Simple defocus laser irradiation to suppress self-absorption in laser-induced breakdown spectroscopy (LIBS)

This study introduces a novel and simple way to suppress the self-absorption effect in laser-induced breakdown spectroscopy (LIBS) by utilizing a defocusing laser irradiation technique. For this purpose, a Nd:YAG laser with a wavelength of 1,064 nm and repetition rate of 10 Hz with energy in the range of 10 mJ–50 mJ was used. The laser irradiation was focused by using a 150-mm-focal-length plano-convex lens onto the sample surface under defocusing of approximately –6 mm. Potassium chloride (KCl) and sodium chloride (NaCl) pellet samples were used to demonstrate this achievement. When the defocus position is adjusted to –6 mm for KCl and NaCl samples, the self-reversal in the emission lines of K I 766.4 nm, K I 769.9 nm, Na I 588.9 nm, and Na I 589.5 nm vanish. Meanwhile, the FWHM values of K I 766.4 and K I 769.9 nm are 0.29 nm and 0.23 nm, respectively, during –6 mm defocus laser irradiation, as opposed to 1.24 nm and 0.86 nm under tight focus laser irradiation. Additionally, this work demonstrates that, when the laser energy is changed between 10 and 50 mJ, no self-reversal occurs in the emission lines when –6 mm defocus laser irradiation is applied. Finally, a linear calibration curve was generated using KCl at a high concentration ranging between K concentrations from 16.6% to 29%. It should be noted that, even at such high K concentrations, the calibration curve is still linear. This means that self-absorption is almost negligible. This simple change in defocus laser irradiation will undoubtedly contribute to the suppression of the self-absorption phenomenon, which disrupts LIBS analytical results.

Potassium supply promotes the mitigation of NaCl-induced effects on leaf photochemistry, metabolism and morphology of Setaria viridis

Soil salinity has the potential to severely affect crop performance. To maintain cell functioning and improve salt tolerance, the maintenance of K+ homeostasis is crucial in several plant metabolism processes. Besides, potassium fertilization can efficiently alleviate the perilous effects of salinity. We characterized impacts in Setaria viridis exposed to NaCl and KCl to underlying photochemistry mechanisms, K+ and Na+ shoot contents, enzymatic activity, electrolytic leakage, and morphological responses focusing on non-stomatal limitation of photosynthesis. Plants were exposed to sodium chloride (NaCl; 0, 150 and 250 mM) and potassium chloride (KCl; 0, 5, 9 mM). The exposure to NaCl affected S. viridis leaves morphological and physiologically. Plants submitted to 150 mM showed reductions in performance indexes (PIabs and PItotal; JIP-test), and the presence of positive K- and L-bands. Plants exposed to 250 mM exhibited blockage in electron flow further than QA within 48h and permanent photoinhibition at 96 h. The presence of 9 and 5 mM of KCl counteracted the effects of NaCl on plants submitted to 150 mM, concomitant with increases in K+ accumulation and cell turgidity conservation, causing positive effects in plant growth and metabolism. Neither KCl concentrations were effective in reducing NaCl-induced effects on plants exposed to 250 mM of NaCl. Our results support the conclusion that greater availability of K+ alleviates the harmful effects of salinity in S. viridis under moderate stress and that application of KCl as means of lightning saline stress has a concentration and a salt level limit that must be experimentally determined.

Effects of varying concentrations of different salts on mineral composition of leaves and roots of three pomegranate (Punica granatum L.) cultivars

The present investigation assesses the behavior of three pomegranate cultivars (Punica granatum L.) (‘Wonderful’, ‘Ermioni’ and ‘Grenada’) under increased concentrations of three salts (NaCl, KCl and K2SO4) after 150 days of growth in vivo conditions. The aim of this research was to study the nutrient accumulation and distribution in leaves and roots and its effects on total chlorophyll (a + b), carotenoids, photosynthetic rate and stomatal conductance of leaves. In general, high salt supply of KCl and K2SO4 led to significant decline in Ca and Mg concentration in leaves in all cultivars. ‘Grenada’ found with the highest concentration of Mn in leaves under all salt treatments. Regarding the roots, ‘Wonderful’ had the highest Mg concentration at 25, 50 and 75 mM of KCl and K2SO4, while ‘Ermioni’ had the maximum Ca concentration in roots. Chlorophyll concentration was reduced by the effect of KCl and K2SO4 salts in all cultivars. Furthermore, K2SO4 salt significantly reduced carotenoid concentration in leaves in all cultivars, especially ‘Wonderful’ showed 63.36 % reduction. ‘Grenada’ had the lowest photosynthetic rate and K2SO4 salt exerted a significant effect in all cultivars. Regarding stomatal conductance, ‘Ermioni’ had the highest values with KCl and K2SO4 salt treatments, while ‘Grenada’ was the most affected cultivar by all salts and at all concentrations. Our findings show the considerable potential of exploiting saline soils to obtain pomegranate plants with higher level of salt tolerance. Furthermore, our results suggest that ‘Wonderful’ and ‘Ermioni’ pomegranate cultivars may be cultivated also under marginal saline conditions.

Synergistic effect of KCl, thiourea, GA3 and SA on the germination and early seedling growth enhancement of drought-stressed Malaysian indica rice cv. MR220

Abstract Rice plantation is one of the sectors that severely affected by drought stress worldwide and caused high loss of rice production. Latest, approximately 1164 ha rice plantation was affected due to water scarcity in Malaysia and reduced rice production. This study suggest that treatment by using liquid formulation containing macronutrient and plant growth regulators as one of the strategies to alleviate drought stress in plant growth and development. Hence, this study aimed to assess the synergistic effect of KCl, thiourea (TU), GA3 and SA for the germination and early growth enhancement of drought-stressed rice cv. MR220. MR220 seeds were stressed in -1.2 MPa PEG 6000 solution and germinated in petri dishes containing KCl, TU, GA3 and SA separately for ten days. Results showed that, 1.0 mM of KCl, 2.0 mM of TU, 0.06 mM of GA3 and 1.0 mM of SA were the ideal concentration that enhance the germination and early seedling growth of drought-stressed MR220. Next, the ideal concentrations were combined and the result showed that the best combination treatments for drought-stressed MR220 were 1.0 mM KCl +0.06 mM GA3, 0.06 mM GA3 + 1.0 mM SA and 1.0 mM KCl +2.0 mM TU + 0.06 mM GA3. These combination treatments significantly increased the germination and seedling growth performance of drought-stressed MR220. The combination treatment of 1.0 mM KCl +2.0 mM TU + 0.06 mM of GA3 showed the most effective in improving germination and seedling growth performance of drought-stressed MR220.

Redefining operant conditioning of escape behaviour in Lymnaea stagnalis

The escape behaviour is one of the many behavioural responses that can be operantly conditioned in a stimulus-dependent manner in both vertebrates and invertebrates. By exposing the pond snail Lymnaea stagnalis repeatedly to a negative reinforcement its natural tendency to explore its surroundings can be operantly conditioned in both adult and aged snails. When adult snails were trained with 100 mM of KCl their number of escapes was significantly decreased and the latency to first escape was significantly increased. Our behavioural protocol allowed us to investigate memory acquisition, consolidation, and retrieval in pre- and post-training sessions over different days. From the 3rd day of training the learned response was strengthened: the number of the escapes in the post-test session remained significantly reduced even when animals were presented with distilled water. Moreover, adult snails exposed to the negative reinforcement for at least 4 days started to escape significantly less than the control group also in the pre-test session. This effect became more pronounced in the following days and was accompanied by a significant increase in the latency to first escape at the beginning of the pre-test on day 6 and 7. Aged snails, instead, showed selective deficiencies when operantly conditioned: memory retention appeared only after 7 days, while memory retrieval could not be induced. This redefined paradigm can help unravelling a variety of sophisticated cognitive phenomena in L. stagnalis and could be employed also to study the basis of memory impairment occurring during neuro-aging.

Effects of Physical and Chemical Stressors on DNA Conformation Investigated By Temperature-Controlled Electrochemical Microdevices.

Effects of Physical and Chemical Stressors on DNA Conformation Investigated By Temperature-Controlled Electrochemical Microdevices.

Removal of arsenite from aqueous solutions using ultrasonic irradiation in the presence of a lead electrode

In this study, we performed electrochemical reduction of H2O2 (H2O2 + e− = OH· + OH−, H2O2 + 2e− = 2OH−) that was generated by ultrasound irradiation and oxidation of As(III) using OH· and H2O2. Electrochemical reduction was conducted using a Pb electrode, which was immersed in 10 ppm of As(III) solution containing 20 mM of KCl and 10 ppm of H2O2, and applied electrical potential of −0.5 V versus Ag/AgCl for 30 min in Ar atmosphere. After electrochemical reduction, the pH increased from 4.8 to 9.3. The concentration of As(V), which was oxidized from As(III) by H2O2, increased from 0 to 6.2 ppm. The precipitate of Pb, β-PbO, and Pb5O8 was obtained by ultrasound irradiation in the presence of the Pb electrode at −0.5 V, and the concentration of As deceased from 10 to 1.2 ppm. The precipitate adsorbed As(V) after oxidation of As(III) by OH· and H2O2.

Development of Liquid Enhancer for Germination of Drought-stressed Oryza sativa subsp. indica Seed cv. MR284

Aim: This study was conducted to develop liquid enhancer containing KCl, TU, GA, and SA for germination of drought-stressed Oryza sativa subsp. indica cv. MR284 seed.
 Study Design: All experiments were conducted in a completely randomized design. Two steps were involved in the development process which are to select an ideal concentration for each KCl, TU, GA, and SA, and to find an ideal combination of chemicals from the selection of ideal concentrations acquired in step 1 to form liquid enhancer. There were 20 treatments for step 1 and 9 treatments for step 2. All of these treatments with 6 replicates.
 Place and Duration of Study: Department of Biology, Faculty of Science, University Putra Malaysia, between June 2018 and December 2018.
 Methodology: The sterilized rice seed cv. MR284 was stressed in the -1.2 Mpa PEG 6000 solution for three days and germinated in the KCl, TU, GA, and SA solution in a series of concentration for 10 days, in a controlled room. Seed germination was observed daily.
 Results: In the first step, drought-stressed rice seed showed the best germination performance in the 30 mM of KCl, 2.0 mM of TU, 0.24 mM GA, and 0.5 mM SA. Meanwhile, in the second step, the drought-stressed rice seed showed the best germination performance in the combination of 30 mM KCl + 2.0 mM TU + 0.24 mM GA + 0.5 mM SA. The best germination performance was evaluated by the highest germination percentage (%), germination index, seed vigor, leaf length, root length and biomass.
 Conclusion: Therefore, the combination treatments of 30 mM KCl + 2.0 mM TU + 0.5 mM SA was found to be the most effective and simplest liquid enhancer formula that has an ability to enhance seed germination of drought-stressed rice cv. MR284 seed.

2172-P: Effects of Glucocorticoid on Gene Expression and Secretion of Insulin in Pancreatic ß Cells

Glucocorticoids are widely used in clinical practice, while the chronic administration at a high dose often leads to the progression of steroid-induced diabetes. This hyperglycemic state results mainly from the suppression of insulin secretion due to pancreatic β-cell dysfunction and the increment of systemic insulin resistance. However, the mechanisms which underlie glucocorticoid action on expression and secretion of insulin in pancreatic β-cells have not been clarified. The aim of this study is to elucidate the effects of glucocorticoid on insulin synthesis and secretion in pancreatic β-cells. Insulin gene expression was significantly increased in 30nM of dexamethasone and decreased in 300 and 3000 nM of dexamethasone. These changes in the expression were canceled by RU486, a GR antagonist, at any concentrations of dexamethasone. Meanwhile, insulin and proinsulin secretion were significantly reduced at any concentrations of dexamethasone. Interestingly, the proinsulin/insulin ratio was significantly decreased at 30 nM, and increased at 300 and 3000 nM. The changes were completely restored by RU486 at 30 nM of dexamethasone, while the reduction of insulin and proinsulin secretion by 300 or 3000 nM of dexamethasone was not released by RU486. In addition, similar results were obtained in experiments using knockdown of GR expression. Interestingly, the short-term induction of insulin secretion by membrane depolarization treated with 30 mM of KCl was rather enhanced by 3000 nM of dexamethasone, and this enhancement was not canceled by RU486. In conclusion, glucocorticoid regulates insulin gene expressions via the genomic pathway containing GR in pancreatic β-cells, while particularly high-dose glucocorticoid regulates insulin secretion principally via non-genomic pathway. Since the proinsulin/insulin ratio increased under chronic administration of high-dose glucocorticoid, it was assumed that the non-genomic pathway was involved in insulin processing. Disclosure T. Murashima: None. T. Kondo: None. K. Kondo: None. N. Morita: None. A. Kitahara: None. Y. Sumitani: None. K. Takahashi: None. T. Tanaka: None. T. Hosaka: Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Ono Pharmaceutical Co., Ltd. H. Ishida: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim International GmbH, Eli Lilly and Company, Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Kowa pharmaceutical Co. Ltd., MSD, Ono Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho. Funding Japan Ministry of Education, Culture, Sports, Science and Technology

Cannabinoids Induce Cell Death and Promote P2X7 Receptor Signaling in Retinal Glial Progenitors in Culture.

Development of progenitors in the embryonic retina is modulated by signaling molecules, and cannabinoid receptors are highly expressed in the early developing retina. Here, we investigated whether the CB1/CB2 receptor agonist WIN 5212-2 (WIN) modulated the proliferation, viability, and calcium responses in chick embryo retinal progenitors in culture. A decline in [3H]-thymidine incorporation was observed when cultures were incubated with 0.5-1.0μM WIN, an effect that was mimicked by URB602 and URB597, inhibitors of the monoacylglycerol lipase and fatty acid amide hydrolase, respectively. A reduction in the number of proliferating cell nuclear antigen-positive nuclei was also noticed in WIN-treated cultures, suggesting that activation of cannabinoid receptors decreases the proliferation of cultured retinal progenitors. WIN (0.5-5.0μM), but not capsaicin, decreased retinal cell viability, an effect that was blocked by CB1 and CB2 receptor antagonists and by the P2X7 receptor antagonist A438079, implicating this nucleotide receptor in the cannabinoid-mediated cell death. Treatment with WIN also induced an increase in mitochondrial superoxide and P2X7 receptor-mediated uptake of sulforhodamine B in the cultured cells. While a high proportion of cultured cells responded to glutamate, GABA, and 50mM KCl with intracellular calcium shifts, very few cells responded to the activation of P2X7 receptors by ATP. Noteworthy, while decreasing the number of cells responding to glutamate, GABA, and KCl, treatment of the cultures with WIN induced a significant increase in the number of cells responding to 1mM ATP, suggesting that activation of cannabinoid receptors primes P2X7 receptor calcium signaling in retinal progenitors in culture.

English

Spermidine (Spd) is one of the most important polyamines (PAs) showing important roles in growth, development and stress responses in plants. The role of Spd in regulation of a dominant glutathione S-transferase (GST, E.C.2.5.1.18) and ionic balance of Na+ and K+ in onion leaves were examined. Onion GSTs were separated from fresh bulb tissue using DEAE-cellulose chromatography. Three GSTs were eluted at 56, 120 and 169 mM of KCl. Among them, GST2 containing >60% of total activity was termed as dominant GST and it was subsequently purified using affinity chromatography S-hexylglutathione-agarose. This purified protein was used for western blotting analysis of the GST in leaves of two months onion seedlings imposed on NaCl induced 16 dSm-1 salinity with or without foliar spray of 100 µM Spd on 1st, 3rd, 5th and 7th days of stress. The results suggested that dominant GST accumulated by salinity in onion seedling with or without Spd spray. However, at early saline stress, accumulation was significantly stronger in leaves without Spd than in those with Spd. At 7th day, GST band was increased and apparently seemed to be intensified in Spd sprayed leaves than those without spray. The values of Na+/K+ suggested that Spd maintained ionic balance better at early stage of stress than late stage.   Key words: Onion glutathione S-transferases (GST), ionic balance, salt stress, spermidine.

Spreading depolarizations trigger caveolin-1-dependent endothelial transcytosis.

Cortical spreading depolarizations (CSDs) are intense and ubiquitous depolarization waves relevant for the pathophysiology of migraine and brain injury. CSDs disrupt the blood-brain barrier (BBB), but the mechanisms are unknown. A total of six CSDs were evoked over 1 hour by topical application of 300 mM of KCl or optogenetically with 470 nm (blue) LED over the right hemisphere in anesthetized mice (C57BL/6 J wild type, Thy1-ChR2-YFP line 18, and cav-1-/- ). BBB disruption was assessed by Evans blue (2% EB, 3 ml/kg, intra-arterial) or dextran (200 mg/kg, fluorescein, 70,000 MW, intra-arterial) extravasation in parietotemporal cortex at 3 to 24 hours after CSD. Endothelial cell ultrastructure was examined using transmission electron microscopy 0 to 24 hours after the same CSD protocol in order to assess vesicular trafficking, endothelial tight junctions, and pericyte integrity. Mice were treated with vehicle, isoform nonselective rho-associated kinase (ROCK) inhibitor fasudil (10 mg/kg, intraperitoneally 30 minutes before CSD), or ROCK-2 selective inhibitor KD025 (200 mg/kg, per oral twice-daily for 5 doses before CSD). We show that CSD-induced BBB opening to water and large molecules is mediated by increased endothelial transcytosis starting between 3 and 6 hours and lasting approximately 24 hours. Endothelial tight junctions, pericytes, and basement membrane remain preserved after CSDs. Moreover, we show that CSD-induced BBB disruption is exclusively caveolin-1-dependent and requires rho-kinase 2 activity. Importantly, hyperoxia failed to prevent CSD-induced BBB breakdown, suggesting that the latter is independent of tissue hypoxia. Our data elucidate the mechanisms by which CSDs lead to transient BBB disruption, with diagnostic and therapeutic implications for migraine and brain injury.

Characterization of Aptamer BC 007 Substance and Product Using Circular Dichroism and Nuclear Magnetic Resonance Spectroscopy

Possible unwanted folding of biopharmaceuticals during manufacturing and storage has resulted in analysis schemes compared to small molecules that include bioanalytical characterization besides chemical characterization. Whether bioanalytical characterization is required for nucleotide-based drugs, may be decided on a case-by-case basis. Nucleotide-based pharmaceuticals, if chemically synthesized, occupy an intermediate position between small-molecule drugs and biologics. Here, we tested whether a physicochemical characterization of a nucleotide-based drug substance, BC 007, was adequate, using circular dichroism (CD) spectroscopy. Nuclear magnetic resonance confirmed CD data in one experimental setup. BC 007 forms a quadruplex structure under specific external conditions, which was characterized for its stability and structural appearance also after denaturation using CD and nuclear magnetic resonance. The amount of the free energy (ΔG0) involved in quadruplex formation of BC 007 was estimated at +8.7 kJ/mol when dissolved in water and +1.4 kJ/mol in 154 mM NaCl, indicating structural instability under these conditions. However, dissolution of the substance in 5 mM of KCl reduced the ΔG0 to −5.6 kJ/mol due to the stabilizing effect of cations. These results show that positive ΔG0 of quadruplex structure formation in water and aqueous NaCl prevents BC 007 from preforming stable 3-dimensional structures, which could potentially affect drug function.

Direct actions of the flavonoids naringenin, quercetin and genistein on rat cardiac and vascular muscles

Context: Flavonoids are natural polyphenolic compounds that are ubiquitous in plants. Numerous prospective epidemiological studies have found beneficial effects of flavonoid consumption on overall cardiovascular mortality. These effects have been attributed to nonspecific antioxidant, anti-atherosclerotic and antithrombotic properties of flavonoids. However, little is known about direct actions of flavonoids on cardiac and vascular smooth muscles. Aims: To evaluate the effects of three flavonoids (naringenin, quercetin and genistein) on electrical and contractile activities of isolated rat hearts and on contraction of rat abdominal aortic rings. Methods: Surface electrogram (ECG) and force of contraction (FC) were recorded in isolated rat hearts. The ventricular fibrillation threshold was evaluated with the flavonoids. The effects on aortic contraction were assessed in rat abdominal aortic rings pre-contracted with 60 mM of KCl. Results: Genistein tended to prolong the QT interval while naringenin tended to decrease it. The effects of all flavonoids on QRS and RR interval were mild and not statistically significant. Genistein and naringenin produced a negative inotropic effect in isolated rat hearts with an IC50 of 13.8 μM and 33.8 μM, respectively. Quercetin at low concentrations had a positive inotropic effect; at high concentrations, it exerted a negative inotropic effect. Naringenin and quercetin increased the threshold for ventricular fibrillation, whereas genistein decreased it. Naringenin, quercetin, and genistein induced concentration-dependent relaxation in endothelium-denuded rat aortic rings. Conclusions: These flavonoids have direct actions on rat cardiac and vascular smooth muscles.

Risk assessment of bis-ethylhexyloxyphenol methoxyphenyl triazine in cosmetic products

Animal toxins are natural resources for pharmacological studies. The venom of Crotalus durissus cascavella (C.d. cascavella) may be a source in the bio-prospecting of new anti-hypertensive agents. The aim of this study was to investigate vascular effects of the venom of C.d. cascavella in normotensive rats. Studies were performed using isolated mesenteric artery segments and aortic endothelial cells. The cumulative administration of the venom of C.d. cascavella (0.001–30 μg/mL) on phenylephrine (Phe; 10 μM) pre-contracted rings induced a concentration-dependent vasorelaxation in the presence of vascular endothelium (Emax = 47.9 ± 5.0% n = 8), and its effect was almost abolished in the absence of endothelium (Emax = 5.8± 2.4% n = 5 (∗∗∗p < 0.001)). Tissue viability was maintained as there was no difference in the contractile capacity of rings before and after the administration of venom. The vasorelaxant effect of the venom was also abolished when arteries were pre-contracted with potassium chloride (KCl; 80 mM) (Emax = 6.4± 0.9% n = 5, ∗∗∗p < 0.001). When assessing the participation of endothelium-derived relaxing factors, it was noted that non-selective COX inhibition with indomethacin (10 μM) caused a significant reduction in the vasorelaxant effect of C.d. cascavella (*p < 0.05). When investigating the participation of NO released by endothelium, there was a significant reduction of the vasorelaxant effect of venom in rings treated with L-NAME (100 μM; Emax = 17.5± 2.2% n = 6; **p < 0.01). Similar results were noted in the presence of ODQ (10 μM), an inhibitor of soluble guanylyl cyclase (Emax = 11.2± 3.5%, n = 6) and PTIO (100 μM), a stable radical scavenger for nitric oxide (Emax = 10.77± 3.6%, n = 6). Moreover, the venom induced the release of NO by isolated aortic endothelial cells through amperometric studies. When assessing the participation of K+ channels on the vasodilatory response of the venom, tyrode solution with 20 mM of KCl caused a significant reduction in the relaxation response (p < 0.001) (Emax = 21.3 ± 8%, n = 7), as did inhibitor of delayed rectifier K+ channels (4-amynopiridine 1 mM; Emax = 9.5 ± 1.3, %, n = 5, ***p < 0.001), and vasorelaxation was almost abolished in the presence of Iberiotoxin (IbTx 100 nM). Therefore, these results suggest that the venom of C.d. cascavella induces vasorelaxation in superior mesenteric artery rings of normotensive rats in an endothelium-dependent manner. Specifically, the venom stimulates the generation of endothelium-derived relaxing factors, especially NO, and activates vascular smooth muscle hyperpolarization through K+ channels. These data illustrate that C.d. cascavella is a source of bioactive molecules and therefore has therapeutic potential in the treatment of cardiovascular diseases such as hypertension.

NO production and potassium channels activation induced by Crotalus durissus cascavella underlie mesenteric artery relaxation

Animal toxins are natural resources for pharmacological studies. The venom of Crotalus durissus cascavella (C.d. cascavella) may be a source in the bio-prospecting of new anti-hypertensive agents. The aim of this study was to investigate vascular effects of the venom of C.d. cascavella in normotensive rats. Studies were performed using isolated mesenteric artery segments and aortic endothelial cells. The cumulative administration of the venom of C.d. cascavella (0.001–30 μg/mL) on phenylephrine (Phe; 10 μM) pre-contracted rings induced a concentration-dependent vasorelaxation in the presence of vascular endothelium (Emax = 47.9 ± 5.0% n = 8), and its effect was almost abolished in the absence of endothelium (Emax = 5.8± 2.4% n = 5 (∗∗∗p < 0.001)). Tissue viability was maintained as there was no difference in the contractile capacity of rings before and after the administration of venom. The vasorelaxant effect of the venom was also abolished when arteries were pre-contracted with potassium chloride (KCl; 80 mM) (Emax = 6.4± 0.9% n = 5, ∗∗∗p < 0.001). When assessing the participation of endothelium-derived relaxing factors, it was noted that non-selective COX inhibition with indomethacin (10 μM) caused a significant reduction in the vasorelaxant effect of C.d. cascavella (*p < 0.05). When investigating the participation of NO released by endothelium, there was a significant reduction of the vasorelaxant effect of venom in rings treated with L-NAME (100 μM; Emax = 17.5± 2.2% n = 6; **p < 0.01). Similar results were noted in the presence of ODQ (10 μM), an inhibitor of soluble guanylyl cyclase (Emax = 11.2± 3.5%, n = 6) and PTIO (100 μM), a stable radical scavenger for nitric oxide (Emax = 10.77± 3.6%, n = 6). Moreover, the venom induced the release of NO by isolated aortic endothelial cells through amperometric studies. When assessing the participation of K+ channels on the vasodilatory response of the venom, tyrode solution with 20 mM of KCl caused a significant reduction in the relaxation response (p < 0.001) (Emax = 21.3 ± 8%, n = 7), as did inhibitor of delayed rectifier K+ channels (4-amynopiridine 1 mM; Emax = 9.5 ± 1.3, %, n = 5, ***p < 0.001), and vasorelaxation was almost abolished in the presence of Iberiotoxin (IbTx 100 nM). Therefore, these results suggest that the venom of C.d. cascavella induces vasorelaxation in superior mesenteric artery rings of normotensive rats in an endothelium-dependent manner. Specifically, the venom stimulates the generation of endothelium-derived relaxing factors, especially NO, and activates vascular smooth muscle hyperpolarization through K+ channels. These data illustrate that C.d. cascavella is a source of bioactive molecules and therefore has therapeutic potential in the treatment of cardiovascular diseases such as hypertension.

Crocin-Induced Endothelium-Dependent Relaxation in Isolated Rat Aorta

Background: Crocin, an active ingredient of saffron, has been found to possess hypotensive effects. Objectives: The aim of this study was to evaluate the mechanism of vasodilatory effects induced by crocin on isolated rat aorta. Methods: To study the vasodilatory effects of crocin (0.1, 0.2, 0.4 and 0.8 M), isolated rat thoracic aorta rings were contracted by 10-6 M Phenylephrine (PE) or 80 mM of KCl. To assess the role of nitric oxide in mechanism of relaxation, induced by crocin, aortic rings were incubated by L-NG-Nitroarginine methyl ester (L-NAME: 10-6 M) for 20 minutes. Furthermore, the relaxant effect of crocin was evaluated both in intact and denuded endothelium aortic rings. Results: Crocin induced relaxation in endothelium-intact aortic rings precontracted with PE in a concentration dependent manner yet not in KCl precontracted aortic rings. The relaxant activity of crocin was abolished by incubation of aortic rings with L-NAME. In addition, the endothelium removal inhibited the relaxant activity of crocin. Conclusions: This study indicated that relaxant activity of crocin is mainly mediated through an endothelium-dependent mechanism.