Angiotensin II-induced activation of c-Ret signaling is critical in ureteric bud branching morphogenesis

The renin-angiotensin system (RAS) plays a critical role in ureteric bud (UB) and kidney morphogenesis. Mutations in the genes encoding components of the RAS cause a spectrum of congenital abnormalities of the kidney and urinary tract (CAKUT). However, the mechanisms by which aberrations in the RAS result in CAKUT are poorly understood. Given that c-Ret receptor tyrosine kinase (RTK) is a major inducer of UB branching, the present study tested the hypothesis that angiotensin (Ang) II-induced activation of c-Ret plays a critical role in UB branching morphogenesis. E12.5 mice metanephroi were grown for 24 h in the presence or absence of Ang II, Ang II AT₁ receptor (AT₁R) antagonist candesartan, phosphatidylinositol 3-kinase (PI3 K) inhibitor LY294002 or ERK1/2 inhibitor PD98059. Ang II increased the number of UB tips (61 ± 2.4 vs. 45 ± 4.3, p < 0.05) compared with control. Quantitative RT-PCR analysis demonstrated that Ang II increased c-Ret mRNA levels in the kidney (1.35 ± 0.05 vs. 1.0 ± 0, p < 0.01) and in the UB cells (1.28 ± 0.04 vs. 1.0 ± 0, p < 0.01) compared to control. This was accompanied by increased Tyr¹⁰⁶²Ret phosphorylation by Ang II (5.5 ± 0.9 vs. 1.8 ± 0.4 relative units, p < 0.05). In addition, treatment of UB cells with Ang II (10^?5 M) increased phosphorylation of Akt compared to control (213 ± 16 vs. 100 ± 20%, p < 0.05). In contrast, treatment of metanephroi or UB cells with candesartan decreased c-Ret mRNA levels (0.72 ± 0.06 vs. 1.0 ± 0, p < 0.01; 0.68 ± 0.07 vs. 1.0 ± 0, p < 0.05, respectively) compared with control. Ang II-induced UB branching was abrogated by LY294002 (24 ± 2.6 vs. 37 ± 3.0, p < 0.05) or PD98059 (33 ± 2.0 vs. 48 ± 2.2, p < 0.01). These data demonstrate that Ang II-induced UB branching depends on activation of Akt and ERK1/2. We conclude that cross-talk between the RAS and c-Ret signaling plays an important role in the development of the renal collecting system.     

Angiotensin III stimulates high stretch-induced ANP secretion via angiotensin type 2 receptor

Angiotensin III (Ang III) is metabolized from Ang II by aminopeptidase (AP) A and in turn, Ang III is metabolized to Ang IV by APN. Ang III is known to have a similar effect to Ang II on aldosterone secretion, but the effect of Ang III on atrial natriuretic peptide (ANP) secretion from cardiac atria is not known. The aim of the present study is to define the effect of Ang III on ANP secretion and its receptor subtype using isolated perfused beating atria. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH₂O to 7.5 cmH₂O. Atrial stretch by volume load increased atrial contractility and ANP secretion. Ang III stimulated stretch-induced ANP secretion in a dose-dependent manner without change in atrial contractility. The stimulated effect of Ang III (1 μM) on stretch-induced ANP secretion was blocked by the pretreatment of Ang II type 2 (AT₂) receptor antagonist but not by AT₁ or Mas receptor antagonist. Pretreatment with inhibitor of phosphoinositide 3-kinase (PI3K), Akt, nitric oxide synthase, soluble guanylyl cyclase, or protein kinase G (PKG) attenuated Ang III-stimulated ANP secretion. When Ang III (40 nM) or Ang II (4 nM) was infused for 10 min into anesthetized rats, mean arterial pressure was increased about 10%. However, Ang III increased plasma ANP level by 35.81 ± 10.19% but Ang II decreased plasma ANP level by 30.41 ± 7.27%. Therefore, we suggest that Ang III, opposite to Ang II, stimulated stretch-induced ANP secretion through AT₂ receptor/PI3K/Akt/nitric oxide/PKG pathway.     

Atg5 deficiency-mediated mitophagy aggravates cardiac inflammation and injury in response to angiotensin II

ObjectiveHypertension induces end-organ damage through inflammation, and autophagy plays a crucial role in the regulation of cellular homeostasis. In the present study, we aimed to define the role of autophagy in the development of inflammation and cardiac injury induced by angiotensin II (Ang II).Methods and ResultsAutophagy protein 5 (Atg5) haplodeficiency (Atg5^+/−) and age-matched wild-type (WT) C57BL/6 J mice were infused with Ang II (1500 ng/kg/min) or saline for 7 days. Heart sections were stained with hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical stains. Cytokine and LC3 levels were measured using real-time PCR or western blot analysis. After Ang II infusion, the WT mice exhibited marked macrophage accumulation, cytokine expression, and reactive oxygen species (ROS) production compared with saline-infused controls. However, these effects induced by Ang II infusion were aggravated in Atg5^+/− mice. These effects were associated with Atg5-mediated impaired autophagy, accompanied by increased production of ROS and activation of nuclear factor-κB (NF-κB) in macrophages. Finally, increased cardiac inflammation in Atg5 haplodeficient mice was associated with increased cardiac fibrosis.ConclusionAtg5 deficiency-mediated autophagy increases ROS production and NF-κB activity in macrophages, thereby contributing to cardiac inflammation and injury. Thus, improving autophagy may be a novel therapeutic strategy to ameliorate hypertension-induced inflammation and organ damage.     

Oxygen regulates the effective diffusion distance of nitric oxide in the aortic wall

Endothelium-derived nitric oxide (NO) is critical in maintaining vascular tone. Accumulating evidence shows that NO bioavailability is regulated by oxygen concentration. However, it is unclear to what extent the oxygen concentration regulates NO bioavailability in the vascular wall. In this study, a recently developed experimental setup was used to measure the NO diffusion flux across the aortic wall at various oxygen concentrations. It was observed that for a constant NO concentration at the endothelial surface, the measured NO diffusion flux out of the adventitial surface at [O₂] = 0 μM is around fivefold greater than at [O₂] = 150 μM, indicating that NO is consumed in the aortic wall in an oxygen-dependent manner. Analysis of experimental data shows that the rate of NO consumption in the aortic wall is first order with respect to [NO] and first order with respect to [O₂], and the rate constant k₁ was determined as (4.0 ± 0.3) × 10³ M^?1 s^?1. Computer simulations demonstrate that NO concentration distribution significantly changes with oxygen concentration and the effective NO diffusion distance at low oxygen level ([O₂] ≤ 25 μM) is significantly longer than that at high oxygen level ([O₂] = 200 μM). These results suggest that oxygen-dependent NO consumption may play an important role in dilating blood vessels during hypoxia by increasing the effective NO diffusion distance.     

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria

The effect of Ca²⁺ applied in high concentrations (50 and 300 µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H₂O₂ release from mitochondria was induced by Ca²⁺ applied in 50 µM, but not in 300 µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca²⁺. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca²⁺ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca²⁺ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca²⁺ given in 50 µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H₂O₂ was significantly higher than that observed in a medium containing ADP and Ca²⁺ no longer increased but rather decreased the rate of H₂O₂ release. With 300 µM Ca²⁺ only a loss but no tendency of a recovery of ΔΨm was detected and H₂O₂ release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca²⁺ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H₂O₂ release is induced by calcium, but only in ≤ 100 µM concentration, when after a transient Ca²⁺-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca²⁺-induced depolarization, Ca²⁺ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300 µM) are unrelated to mitochondrial permeability transition.     

The effects of angiotensin II and the oxidative stress mediator 4-hydroxynonenal on human osteoblast-like cell growth: possible relevance to otosclerosis

Otosclerosis is a complex disease characterized by an abnormal bone turnover of the otic capsule resulting in conductive hearing loss. Recent findings have shown that angiotensin II (Ang II), a major effector peptide of the renin–angiotensin system, plays an important role in the pathophysiology of otosclerosis, most likely by its proinflammatory effects on the bone cells. Because reactive oxygen species play a role both in inflammation and in the cellular signaling pathway of Ang II, the appearance of protein adducts of the “second messenger of free radicals,” the aldehyde 4-hydroxynonenal (HNE), in otosclerotic bone has been analyzed. Immunohistochemical analysis of HNE-modified proteins in tissue samples of the stapedial bones performed on 15 otosclerotic patients and 6 controls revealed regular HNE–protein adducts present in the subperiosteal parts of control bone specimens, whereas irregular areas of a pronounced HNE–protein adduct presence were found within stapedial bone in cases of otosclerosis. To study possible interference by HNE and Ang II in human bone cell proliferation, differentiation, and induction of apoptosis we used an in vitro model of osteoblast-like cells. HNE interacted with Ang II in a dose-dependent manner, both by forming HNE–Ang II adducts, as revealed by immunoblotting, and by modifying its effects on cultured cells. Namely, treatment with 0.1 nM Ang II and 2.5 μM HNE stimulated proliferation, whereas treatment with 10 μM HNE or in combination with Ang II (0.1, 0.5, and 1 nM) decreased cell proliferation. Moreover, 10 μM HNE alone and with Ang II (except if 1 nM Ang II was used) increased cellular differentiation and apoptosis. HNE at 5 μM did not affect differentiation nor significantly change apoptosis. On the other hand, when cells were treated with lower concentrations of HNE and Ang II we observed a decrease in cellular differentiation (combination of 1.0 or 2.5 μM HNE with 0.1 nM Ang II) and decrease in apoptosis (0.1 and 0.5 nM Ang II). Cellular necrosis was increased with 5 and 10 μM HNE if given alone or combined with Ang II, whereas 0.5 nM Ang II and combination of 1 μM HNE with Ang II (0.1 and 0.5 nM) reduced necrosis. These results indicate that HNE and Ang II might act mutually dependently in the regulation of bone cell growth and in the pathophysiology of otosclerosis.     

Docosahexaenoic acid reverses angiotensin II-induced RECK suppression and cardiac fibroblast migration

The omega-3 polyunsaturated fatty acids (ω − 3 fatty acids) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to inhibit or delay the progression of cardiovascular diseases, including myocardial fibrosis. Recently we reported that angiotensin II (Ang II) promotes cardiac fibroblast (CF) migration by suppressing the MMP regulator reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), through a mechanism dependent on AT1, ERK, and Sp1. Here we investigated the role of miR-21 in Ang II-mediated RECK suppression, and determined whether the ω − 3 fatty acids reverse these effects. Ang II induced miR-21 expression in primary mouse cardiac fibroblasts (CFs) via ERK-dependent AP-1 and STAT3 activation, and while a miR-21 inhibitor reversed Ang II-induced RECK suppression, a miR-21 mimic inhibited both RECK expression and Ang II-induced CF migration. Moreover, Ang II suppressed the pro-apoptotic PTEN, and the ERK negative regulator Sprouty homologue 1 (SPRY1), but induced the metalloendopeptidase MMP2, all in a manner that was miR-21-dependent. Further, forced expression of PTEN inhibited Akt phosphorylation, Sp1 activation, and MMP2 induction. Notably, while both EPA and DHA reversed Ang II-mediated RECK suppression, DHA appeared to be more effective, and reversed Ang II-induced miR-21 expression, RECK suppression, MMP2 induction, and CF migration. These results indicate that Ang II-induced CF migration is differentially regulated by miR-21-mediated MMP induction and RECK suppression, and that DHA has the potential to upregulate RECK, and therefore may exert potential beneficial effects in cardiac fibrosis.     

An amperometric H2O2 biosensor based on cytochrome c immobilized onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode

Cytochrome c was immobilized covalently onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited on gold (Au) electrode. An amperometric H₂O₂ biosensor was constructed by connecting this modified Au electrode along Ag/AgCl as reference and Pt wire as counter electrode to the galvanostat. The modified Au electrode was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR). Cyclic voltammetric (CV) studies of the electrode at different stages demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H₂O₂, which offered a number of attractive features to develop an amperometric biosensor based on split of H₂O₂. There was a good linear relationship between the current (mA) and H₂O₂ concentration in the range 3–700 μM. The sensor had a detection limit of 0.2 μM (S/N = 3) with a high sensitivity of 3.3 mA μM^?1 cm^?2. The sensor gave accurate and satisfactory results, when employed for determination of H₂O₂ in different fruit juices.     

High intensity interval exercise decreases IL-8 and enhances the immunomodulatory cytokine interleukin-10 in lean and overweight–obese individuals

PurposeTo compare the effects of two interval exercises with different intensities on acute inflammatory response in lean and overweight–obese subjects.MethodsTen lean (BMI < 24.9 kg/m²) and 12 overweight–obese (BMI 25 to <34.9 kg/m²) males performed two conditions in randomly assigned: (1) high intensity interval exercise (HIIE) 10 × 60 s (85–90%P_Max)/75 s (50%P_Max); (2) moderate intensity interval exercise (MIIE) 10 × 60 s (70–75%P_Max)/60 s (50%P_Max), with blood collections at pre, immediately and 30 min post each exercise bouts to evaluate total and differential leukocyte counts, serum creatine kinase (CK), lactate dehydrogenase (LDH) and systemic levels of IL-1ra, IL-6, IL-8, IL-10, IL-17a and CCL2.ResultsIn lean group, HIIE induced a significant increase in total leukocytes and monocyte, while MIIE session did not change the number of leukocytes. Overweight–obese group presented similar increase in leukocytes, monocytes and lymphocytes in both HIIE and MIIE sessions. At baseline, overweight–obese group showed high levels of CK, IL-8, IL-6 and CCL2 and lower concentrations of IL-10 compared to lean group. The MIIE did not alter the cytokine concentrations in both groups, independently of the time analysis. The HIIE induced significant decrease in IL-8 levels 30 min post session in both the groups, and a progressive elevation in IL-10 levels immediately and 30 min post in lean and overweight–obese. Regarding IL-6, overweight–obese subjects presented progressive increase either immediately and 30 min after HIIE, while lean individuals presented significant increase only 30 min after exercise.ConclusionsThe acute inflammatory response to interval exercise is intensity-dependent. Although obesity influences the basal concentrations of several cytokines, only HIIE induced important alterations in IL-8 and IL-10 levels, which may have important implications in the control of chronic low-grade inflammation in obesity.     

Microbial transformation of β-lapachone to its glycosides by Cunninghamella elegans ATCC 10028b

β-lapachone (1) has entered phases I and II clinical trials for the treatment of solid tumors and the therapeutic efficacy of β-lapachone is closely related to its metabolic process. In order to contribute to a better understanding of human metabolism of β-lapachone, Cunninghamella elegans ATCC 10028b was used as a microbial model of mammalian metabolism to biotransform β-lapachone and two new glycosylated derivatives were produced. The chemical structures were elucidated as 6-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-5-O-β-d-glucopyranoside (2) and 5-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-6-O-β-d-glucopyranoside (3) by ¹H NMR, ¹³C NMR, HMBC, HMQC, COSY and HRMS analyses. The major derivative (3) displayed a lower activity against breast cancer cell line SKBR-3 (IC₅₀ = 312.5 μM) than β-lapachone (IC₅₀ = 5.6 μM), but did not show cytotoxicity against normal fibroblasts cell line GM07492-A, whereas β-lapachone was highly toxic (IC₅₀ = 7.25 μM). These metabolites were reported here for the first time and are similar to those that occur in phase II of human metabolism     

The effect of PI3 kinase inhibitor LY294002 on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

AimsWe examined the effect of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on voltage-dependent K⁺ (Kv) channels.Main methodsElectrophysiological recordings were performed in freshly isolated rabbit coronary arterial smooth muscle cells.Key findingsThe Kv current amplitude was inhibited by LY294002 in a dose-dependent manner, with a K_d value of 1.48 μM. Without alteration of the kinetics of activation, LY294002 accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for LY294002 were 1.83 ± 0.01 μM^? 1 s^? 1 and 2.59 ± 0.14 s^? 1, respectively. Application of LY294002 had no significant impact on the steady-state activation or inactivation curves. In the presence of LY294002, the recovery time constant from inactivation was increased, and Kv channel inhibition increased under train pulses (1 or 2 Hz). This indicates that LY294002-induced Kv channel inhibition is use-dependent. Furthermore, pretreatment with another PI3K inhibitor, wortmannin (10 μM), did not affect the Kv current, and did not change the inhibitory effect of LY294002.SignificanceBased on these results, we suggest that LY294002 directly blocks Kv current irrespective of PI3K inhibition.     

Impact of hydrogen peroxide-driven Fenton reaction on mouse oocyte quality

Here we show that hydroxyl radical ^(•OH) generated through the Fenton reaction alters metaphase-II mouse oocyte microtubules (MT) and chromosomal alignment (CH). Metaphase-II mouse oocytes, obtained commercially, were grouped as follows: control, hydrogen peroxide (H₂O₂), Fe(II), and combined (Fe(II) +H₂O₂) treatments. After 7–10 min of incubation at 37 °C, MT and CH were evaluated on fixed and stained oocytes and scored by two blinded observers. Pearson χ² test and Fisher exact test were used to compare outcomes between controls and treated groups and also among the treated groups. Our results showed that poor scores for MT and CH increased significantly in oocytes treated with a combination of H₂O₂ and Fe(II) (p<0.001); oocytes treated with H₂O₂ alone or Fe(II) alone showed no or few changes compared to control. Comparison of oocyte groups that received increasing concentrations of H₂O₂ and a fixed amount of Fe(II) showed that 70–80% demonstrated poor scores in both MT and CH when pretreated with 5 μM H₂O₂, and this increased up to 90–100% when treated with 10–20 μM H₂O₂. Hydroxyl radical generated by H₂O₂-driven Fenton reaction deteriorates the metaphase-II mouse oocyte spindle and CH alignment, which is thought to be a potential cause of poor oocyte quality. Thus, free iron and/or ROS scavengers could attenuate the ^•OH-mediated spindle and chromosomal damage, thereby serving as a possible approach for further examination as a therapeutic option in inflammatory states.     

Treatment of dye (Remazol Blue) and heavy metals using yeast cells with the purpose of managing polluted textile wastewaters

The bioaccumulation of chromium(VI), nickel(II), copper(II), and reactive dye by the yeast Rhodotorula mucilaginosa has been investigated in media containing molasses as a carbon and energy source. Optimal pH values for the yeast cells to remove the pollutants were pH 4 for copper(II) and dye, pH 6 for chromium(VI) and dye, and pH 5 for nickel(II) and dye in media containing 50 mg l^?1 heavy metal and 50 mg l^?1 Remazol Blue. The maximum dye bioaccumulation was observed within 4–6 days and uptake yields varied from 93% to 97%. The highest copper(II) removal yields measured were 30.6% for 45.4 mg l^?1 and 32.4% for 95.9 mg l^?1 initial copper(II) concentrations. The nickel(II) removal yield was 45.5% for 22.3 mg l^?1, 38.0% for 34.7 mg l^?1, and 30.3% for 62.2 mg l^?1. Higher chromium(VI) removal yields were obtained, such as 94.5% for 49.2 mg l^?1 and 87.7% for 129.2 mg l^?1 initial chromium(VI) concentration. The maximum dye and heavy metal bioaccumulation yield was investigated in media with a constant dye (approximately 50 mg l^?1) and increasing heavy metal concentration. In the medium with 48.9–98.8 mg l^?1 copper(II) and constant dye concentration, the maximum copper(II) bioaccumulation was 27.7% and 27.9% whereas the maximum dye bioaccumulation was 96.1% and 95.3%. The maximum chromium(VI) bioaccumulation in the medium with dye was 95.2% and 80.3% at 48.2 and 102.2 mg l^?1 chromium(VI) concentrations. In these media dye bioaccumulation was 76.1% and 35.1%, respectively. The highest nickel(II) removal was 6.1%, 20.3% and 16.0% in the medium with 23.8 mg l^?1 nickel(II) + 37.8 mg l^?1 dye, 38.1 mg l^?1 nickel(II) + 33.4 mg l^?1 dye and 59.0 mg l^?1 nickel(II) + 39.2 mg l^?1 dye, respectively. The maximum dye bioaccumulation yield in the media with nickel(II) was 94.1%, 78.0% and 58.7%, respectively.     

Esculentin-2CHa: A host-defense peptide with differential cytotoxicity against bacteria,erythrocytes and tumor cells

The host-defense peptide, esculentin-2CHa (GFSSIFRGVA¹⁰KFASKGLGK D²⁰LAKLGVDLVA³⁰ CKISKQC) shows potent (MIC ≤ 6 μM) growth inhibitory activity against clinical isolates of multidrug-resistant strains of Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia and differential cytotoxic activity against human erythrocytes (LC₅₀ = 150 μM) and human non-small cell lung adenocarcinoma A549 cells (LC₅₀ = 10 μM). Esculentin-2CHa significantly (P < 0.01) stimulates the release of the anti-inflammatory cytokine IL-10 by mouse lymphoid cells and elevates its production after stimulation with concanavalin A and significantly (P < 0.05) stimulates TNF-α production by peritoneal macrophages. Effects on IL-6 and IL-1β production were not significant. Removal of the hydrophobic N-terminal hexapeptide (GFSSIF) from esculentin-2CHa results in abolition of growth inhibitory activity against S. aureus and cytotoxic activity against erythrocytes and A549 cells as well as a marked (≥16-fold) reduction in potency against A. baumannii and S. maltophilia. The primary structure of esculentin-2 has been poorly conserved between frog species but evolutionary pressure has acted to maintain the hydrophobic character of this N-terminal hexapeptide sequence. Removal of the cyclic C-terminal domain (CKISKQC) and replacement of the Cys³¹ and Cys³⁷ residues by serine resulted in appreciable decreases in cytotoxicity against all microorganisms and against mammalian cells. The more cationic [D20K, D27K] analog showed a modest increase in potency against all microorganisms (up to 4-fold) but a marked increase in cytotoxicity against erythrocytes (LC₅₀ = 11 μM) and A549 cells (LC₅₀ = 3 μM).     

Comparison and relationship of thyroid hormones,IL-6, IL-10 and albumin as mortality predictors in case-mix critically ill patients

ObjectiveTo compare the ability of thyroid hormones, IL-6, IL-10, and albumin to predict mortality, and to assess their relationship in case-mix acute critically ill patients.MethodsAPACHE II scores and serum thyroid hormones (FT3, FT4, and TSH), IL-6, IL-10, and albumin were obtained at EICU admission for 79 cases of mix acute critically ill patients without previous history of thyroid disease. Patients were followed for 28 days with patient’s death as the primary outcome. All mean values were compared, correlations assessed with Pearson’ test, and mortality prediction assessed by multivariate logistic regression and ROC.ResultsNon survivors were older, with higher APACHE II score (p = 0.000), IL-6 (p < 0.05), IL-10 (p = 0.000) levels, and lower albumin (p = 0.000) levels compared to survivors at 28 days. IL-6 and IL-10 had significant negative correlation with albumin (p = 0.001) and FT3 (p ⩽ 0.05) respectively, while low albumin had a direct correlation with FT3 (p < 0.05). In the mortality prediction assessment, IL-10, albumin and APACHE II were independent morality predictors and showed to have a good (0.70–0.79) AUC-ROC (p < 0.05). Despite that the entire cohort showed low FT3 serum levels (p = 0.000), there was not statistical difference between survivors and non-survivors; neither showed any significance as mortality predictor.ConclusionsIL-6 and IL-10 are correlated with Low FT3 and hypoalbuminemia. Thyroid hormones assessed at EICU admission did not have any predictive value in our study. And finally, high levels of IL-6 and IL-10 in conjunction with albumin could improve our ability to evaluate disease’s severity and predict mortality in the critically ill patients. When use in combination with APACHE II scores, our model showed improved mortality prediction.     

Fructanolytic and saccharolytic enzymes of Treponema zioleckii strain kT

Enzymes in the newly described rumen bacterium, Treponema zioleckii strain kT, capable of digesting Timothy grass fructan, inulin, and sucrose were identified and characterized. Two specific endolevanases and one non-specific β-fructofuranosidase were found in a cell-free extract. The molecular weight of the endolevanases were estimated to be 60 and 36 kDa, whereas that of β-fructofuranosidase, 87 kDa. The former of the specific enzymes was associated with the outer membrane, while the latter and the non-specific β-fructofuranosidase, with the periplasm or cytosol. The K_m and V_max for Timothy grass fructan degradation by endolevanase were 0.27% and 15.75 μM fructose equivalents × mg protein^?1 × min^?1, those for sucrose and inulin digestion by β-fructofuranosidase were 1.35 × 10^?3 M and 1.73 μM hexoses × mg protein^?1 × min^?1 and 1.77% and 1.83 μM hexoses × mg protein^?1 × min^?1, respectively.     

Acute effects of cocaine,morphine and their combination on bioenergetic function and susceptibility to oxidative stress of rat liver mitochondria

AimsCocaine and heroin are frequently co-abused in a combination known as speedball. Despite the relevance of the liver in the metabolism and detoxification of these drugs, little is known about the impact of speedball on liver function.Main methodsIn this work, we evaluated the effects of cocaine, morphine and morphine + cocaine (Mor + Coc) combination (1:1) in isolated rat liver mitochondria, upon glutamate/malate or succinate energization, on bioenergetics and oxidative stress-related parameters by using Clark O₂, Ca^2 +, TPP⁺ and pH electrodes and by measuring thiobarbituric acid reactive substances (TBARS) and H₂O₂ production.Key findingsCocaine and Mor + Coc at the higher concentrations (1 mM) similarly increased O₂ consumption at state 2, state 4 and state oligomycin. In these conditions, maximum respiration was decreased only upon glutamate/malate energization, suggesting an involvement of complex I. Morphine (1 mM) only increased state 2 respiration. Cocaine and Mor + Coc induced a similar decrease in maximum mitochondrial membrane potential and in ADP-induced depolarization, whereas morphine had no effect. The drugs and their combination similarly decreased mitochondrial ATPase activity and had no effect on Ca^2 +-induced permeability transition. Morphine and Mor + Coc prevented lipid peroxidation, since in these conditions there was a decrease in O₂ consumption and in TBARS upon ADP/Fe^2 + stimulus, and a decrease in H₂O₂ formation, suggesting an antioxidant effect. Interestingly, heroin did not share morphine antioxidant properties.SignificanceOur results show that the sequential direct exposure of liver mitochondria to morphine and cocaine does not alter the effects observed in the presence of each drug alone.     

Effect of 3,4-ethylenedioxy-extension of thiophene core on the DNA/RNA binding properties and biological activity of bisbenzimidazole amidines

Novel bisbenzimidazoles (4–6), characterized by 3,4-ethylenedioxy-extension of thiophene core, revealed pronounced affinity and strong thermal stabilization effect toward ds-DNA. They interact within ds-DNA grooves as dimmers or even oligomers and agglomerate along ds-RNA. Compounds 4–6 have shown moderate to strong antiproliferative effect toward panel of eight carcinoma cell lines. Compound 5 displayed the best inhibitory potential and in equitoxic concentration (IC₅₀ = 1 × 10^?6 M) induced accumulation of cells in G2/M phase after 48 h of incubation. Fluorescence microscopy showed that 5 entered into live HeLa cells within 30 min, but did not accumulate in nuclei even after 2.5 h. Compound 5 inhibited the growth of Trypanosome cruzi epimastigotes (IC₅₀ = 4.3 × 10^?6 M).     

Development of biosensor for phenolic compounds containing PPO in β-cyclodextrin modified support and iridium nanoparticles

A biosensor based on the iridium nanoparticles dispersed in ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate (Ir-BMI·PF₆) and a celery (Apium graveolens) extract as a source of polyphenol oxidase (PPO) was constructed. A modified support based on β-cyclodextrin (β-CDEP) was used for enzyme immobilization. The behavior of phenolic compounds was investigated by square-wave voltammetry and rutin was selected by presenting the greatest signal. The best performance was obtained with a composition of 70:10:10:10% (w/w/w/w) of the graphite powder:β-CDEP:Nujol:Ir-BMI·PF₆ composition, a PPO concentration of 500 units mL^?1, in 0.1 M phosphate buffer solution (pH 6.0) with frequency, pulse amplitude and scan increment at 100 Hz, 60 mV, and 3.0 mV, respectively. Under optimized conditions, the cathodic currents increased linearly for the rutin concentration range of 1.3 × 10^?7–2.0 × 10^?6 M with a detection limit of 7.9 × 10^?8 M. This sensor demonstrated acceptable repeatability and reproducibility and the results for the rutin recovery ranged from 92.8 to 103.4%. A relative error of 0.7% was obtained in the rutin determination in simulated samples.