Erythrocyte membrane AChE,Na+, K+-ATPase and Mg2+ ATPase activities in mothers and their premature neonates in relation to the mode of delivery

Abstract

Aim. To investigate erythrocyte membrane AChE, Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in mothers and their full-term or premature newborns in relation to the mode of delivery. Methods. Blood was obtained from mothers pre- and post-delivery and the umbilical cord (CB) of their full-term newborns: Group A₁ (n = 16) born with vaginal delivery (VD), Group B₁ (n = 14) full-terms with scheduled cesarean section (CS), Group A₂ (n = 12) prematures with VD, Group B₂ (n = 14) prematures with CS. Total Antioxidant Status (TAS) and common laboratory tests were measured with routine methods, and the membrane enzyme activities spectrophotometrically. Results. TAS was reduced in mothers post VD and in the CB whereas remained unaltered in CS mothers and their newborns. AChE and Na⁺, K⁺-ATPase were increased in mothers post VD. AChE was lower in the CB of prematures than that of full-terms independently of the mode of delivery. Na⁺, K⁺-ATPase activity was increased in the groups of mothers post VD and decreased in prematures. The enzyme was higher in prematures with CS than that with VD. Mg²⁺-ATPase activity was unchanged. Conclusion. The increased maternal AChE and Na⁺, K⁺-ATPase activities may be due to the low TAS determined post VD, whereas their decreased activities in prematures to their immaturity.     

Functional correlation of ATP1A2 mutations with phenotypic spectrum: from pure hemiplegic migraine to its variant forms

BackgroundMutations in ATP1A2, the gene encoding the α2 subunit of Na⁺/K⁺-ATPase, are the main cause of familial hemiplegic migraine type 2 (FHM2). The clinical presentation of FHM2 with mutations in the same gene varies from pure FHM to severe forms with epilepsy and intellectual disability, but the correlation of these symptoms with different ATP1A2 mutations is still unclear.MethodsTen ATP1A2 missense mutations were selected according to different phenotypes of FHM patients. They caused pure FHM (FHM: R65W, R202Q, R593W, G762S), FHM with epilepsy (FHME: R548C, E825K, R938P), or FHM with epilepsy and intellectual disability (FHMEI: T378N, G615R, D718N). After ouabain resistance and fluorescence modification, plasmids carrying those mutations were transiently transfected into HEK293T and HeLa cells. The biochemical functions were studied including cell survival assays, membrane protein extraction, western blotting, and Na⁺/K⁺-ATPase activity tests. The electrophysiological functions of G762S, R938P, and G615R mutations were investigated in HEK293T cells using whole-cell patch-clamp. Homology modeling was performed to determine the locational distribution of ATP1A2 mutations.ResultsCompared with wild-type pumps, all mutations showed a similar level of protein expression and decreased cell viability in the presence of 1 µM ouabain, and there was no significant difference among the mutant groups. The changes in Na⁺/K⁺-ATPase activity were correlated with the severity of FHM phenotypes. In the presence of 100 µM ouabain, the Na⁺/K⁺-ATPase activity was FHM > FHME > FHMEI. The ouabain-sensitive Na⁺/K⁺-ATPase activity of each mutant was significantly lower than that of the wild-type protein, and there was no significant difference among all mutant groups. Whole-cell voltage-clamp recordings in HEK293T cells showed that the ouabain-sensitive pump currents of G615R were significantly reduced, while those of G762S and R938P were comparable to those of the wild-type strain.ConclusionsATP1A2 mutations cause phenotypes ranging from pure FHM to FHM with epilepsy and intellectual disability due to varying degrees of deficits in biochemical and electrophysiological properties of Na⁺/K⁺-ATPase. Mutations associated with intellectual disability presented with severe impairment of Na⁺/K⁺-ATPase. Whether epilepsy is accompanied, or the type of epilepsy did not seem to affect the degree of impairment of pump function.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10194-021-01309-4.     

Pentagalloylglucose, an antisecretory component of Paeoniae radix, inhibits gastric H+, K(+)-ATPase

We purified a compound with strong inhibitory effect on H+, K(+)-ATPase from Paeoniae radix, which has been used in Japan for the treatment of gastritis and peptic ulcers. The compound was identified as 1,2,3,4,6,-penta-o-galloyl-beta-D-glucose by proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, and fast atomic bombardment mass spectrometry. The IC50 of the compound for H+, K(+)-ATPase was 166 nmol/l. Kinetic analyses indicated that the inhibition of the enzyme by pentagalloylglucose was noncompetitive with respect to K+. Pentagalloylglucose had relatively weak inhibitory effects for Mg(+)-ATPase (IC50: > 10 mumol/l) and Na+, K(+)-ATPase (IC50: 2.7 mumol/l). Pentagalloylglucose also inhibited the accumulation of [14C]aminopyrine in parietal cells that had been isolated from guinea pig stomach and stimulated by 10 mumol/l histamine (IC50: 7.8 mumol/l) and 1 mmol/l dbc-AMP (IC50: 10 mumol/l). These results suggest that pentagalloylglucose is a potent inhibitor of H+, K(+)-ATPase and may be responsible for inhibition of acid secretion by Paeoniae radix.     

The β3 subunit of the Na,K-ATPase mediates variable nociceptive sensitivity in the formalin test

It is widely appreciated that there is significant inter-individual variability in pain sensitivity, yet only a handful of contributing genetic variants have been identified. Computational genetic mapping and quantitative trait locus analysis suggested that variation within the gene coding for the β₃ subunit of the Na⁺,K⁺-ATPase pump (Atp1b3) contributes to inter-strain differences in the early phase formalin pain behavior. Significant strain differences in Atp1b3 gene expression, β₃ protein expression, and biophysical properties of the Na⁺,K⁺ pump in dorsal root ganglia neurons from resistant (A/J) and sensitive (C57BL/6J) mouse strains supported the genetic prediction. Furthermore, in vivo siRNA knockdown of the β₃ subunit produced strain-specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the β₃ subunit of the Na⁺,K⁺-ATPase is a novel determinant of nociceptive sensitivity and further supports the notion that pain variability genes can have very selective effects on individual pain modalities.     

A fast chemiluminescent method for H2O2 measurement in exhaled breath condensate

Background: Breath condensate can give useful information on volatile compounds produced at alveolar level. Actual concentration of H₂O₂ in breath condensate is dependent on its production at alveolar level and on the efficacy of the detoxifying systems, catalase, glutathione peroxidase, etc. Methods: In the present paper, a simple chemiluminescent method for the determination of the H₂O₂ collected in exhaled breath is shown and data of both smokers and nonsmokers volunteers are presented. Results: The chemiluminescent response is linear up to 100 μmol/l H₂O₂. The analytical sensitivity is about 0.01 μmol/l. Most of the nonsmokers have a H₂O₂ content lower than 0.05 μmol/l, while smokers have a content ranging from 0.1 to 0.6 μmol/l.     

Novel chalcone derivatives containing a 1,2,4-triazine moiety: design,synthesis, antibacterial and antiviral activities

A series of novel chalcone derivatives containing the 1,2,4-triazine moiety were synthesized and their structures were confirmed by ¹H NMR, ¹³C NMR and elemental analyses. Antiviral bioassays revealed that most of the compounds exhibited good antiviral activity against tobacco mosaic virus (TMV) at a concentration of 500 μg mL⁻¹. The designated compound 4l was 50% effective in terms of curative and protective activities against TMV with 50% effective concentrations (EC₅₀) of 10.9 and 79.4 μg mL⁻¹, which were better than those of ningnanmycin (81.4 and 82.2 μg mL⁻¹). Microscale thermophoresis (MST) also showed that the binding of compound 4l to coat protein (TMV-CP) yielded a K_d value of 0.275 ± 0.160 μmol L⁻¹, which was better than that of ningnanmycin (0.523 ± 0.250 μmol L⁻¹). At the same time, molecular docking studies for 4l with TMV-CP (PDB code:1EI7) showed that the compound was embedded well in the pocket between the two subunits of TMV-CP. Meanwhile, compound 4a demonstrated excellent antibacterial activities against Ralstonia solanacearum (R. solanacearum), with an EC₅₀ value of 0.1 μg mL⁻¹, which was better than that of thiodiazole-copper (36.1 μg mL⁻¹) and bismerthiazol (49.5 μg mL⁻¹). The compounds act by causing folding and deformation of the bacterial cell membrane as observed using scanning electron microscopy (SEM). The chalcone derivatives thus synthesized could become potential alternative templates for novel antiviral and antibacterial agents.

A series of novel chalcone derivatives containing the 1,2,4-triazine moiety were synthesized and their structures were confirmed by ¹H NMR, ¹³C NMR and elemental analyses.     

Design,synthesis, and bioactivity evaluation of antitumor sorafenib analogues

Malignant tumors are a serious threat to human health and are generally treated with chemical therapy. This chemical therapy uses agents that act on signal transduction pathway mechanism of tumor with good selectivity and low toxicity. Sorafenib is a multikinase target inhibitor with good tumor inhibitory activity and a protein kinase inhibitor. In this research, a novel series of sorafenib analogues and derivatives were designed, synthesized, and evaluated as tumor inhibitors. These compounds used sorafenib as the lead compound and achieved modifications using bioisosteres and the alkyl principle. The in vitro the results showed that compounds 3c, 3d, 3h, 3n, 3r, and 3z had good inhibitory effects on human cervical cancer cells (Hela), while compounds 3t and 3v had good inhibitory effects on human lung cancer cells (H1975 and A549). Among these, compound 3d had an inhibitory activity (IC₅₀) of 0.56 ± 0.04 μmol L⁻¹ against Hela cells (human cervical cancer), the compound 3t had an IC₅₀ of 2.34 ± 0.07 μmol L⁻¹ against H1975 cells (human lung cancer), and compound 3v had an IC₅₀ of 1.35 ± 0.03 μmol L⁻¹ against A549 cells (human lung cancer). The in vivo results showed that these compounds had good antitumor effects and low acute toxicity.

Malignant tumors are a serious threat to human health and are generally treated with chemical therapy.     

CD137 enhances cytotoxicity of CD3CD56 cells and their capacities to induce CD4 Th1 responses

CD137 (4-1BB) is a TNFR superfamily member that mediates the costimulatory signal resulting in T cells and NK cells proliferation and cytokines production, but the effects of CD137 signaling on CD3⁺CD56⁺ cell subpopulation have not been well-documented. The aim of this study was to investigate the effects of CD137 signaling on regulation of CD3⁺CD56⁺ cell function. Anti-CD137 mAb or mouse IgG1 isotype control was added to CIK cell culture to determine the effects of proliferation and anti-tumor effects on CD3⁺CD56⁺ cells. We observed that anti-CD137 mAb could dramatically promote proliferation of CIK cells. And CD137–CIK cells and CD3⁺CD56⁺ cell subpopulation within them possessed higher ability to kill tumor cell line A549. The SCID mice engrafted with A549 cells and treated with CD137–CIK cells have prolonged survival. Further studies revealed that the percentages of CD3⁺CD56⁺ cells were elevated significantly in CD137–CIK cells. The expression of NKG2D was up-regulated on CD3⁺CD56⁺ cells from CD137–CIK cells. The expression of IFN-γ, IL-2 and TNF-α increased significantly whereas the production of TGF-β₁, IL-4 and IL-10 decreased in CD3⁺CD56⁺ cells from CD137–CIK cells. In addition, anti-CD137 mAb can elevate the capacity of CD3⁺CD56⁺ cells to induce CD4⁺ Th1 responses. We further showed that the anti-CD137 mAb also had the same effects on CD3⁺CD56⁺ cells expanded from the PBMCs of patients with NSCLC. We concluded that CD137 signaling could enhance the abilities of CIK cells to kill tumor cells in vitro and in vivo via increasing the proportion of CD3⁺CD56⁺ cells and their cytotoxicity. Furthermore, CD137 signaling can elevate the capacity of CD3⁺CD56⁺ cells to induce CD4⁺ Th1 responses which may enhance their anti-tumor activity indirectly. Taken together, our studies could be considered as valuable in CIK cells-based cancer immunotherapy.     

A vanillin-based copper(ii) metal complex with a DNA-mediated apoptotic activity

Vanillin (vanH) is the major component of vanilla and one of the most widely used flavoring agents. In this work the complex [Cu(phen)(van)₂] was prepared and characterized by structural (X-ray), spectroscopic (IR, UV-Vis, EPR) and electrochemical techniques. This compound showed an octahedral geometry with an unusual arrangement of the vanillin ligands, where the methoxy groups of the vanillinate ions are coordinated opposite to each other. The compound promoted DNA cleavage in the presence of glutathione (GSH) and H₂O₂. At 40 μmol L⁻¹ of complex with GSH (10 mmol L⁻¹), there is a complete cleavage of DNA to nicked form II, while only at 10 μmol L⁻¹ of this complex with H₂O₂ (1 mmol L⁻¹) an extensive cleavage leading to form III took place. Additionally, we have evidences of superoxide generation upon reaction with GSH. Therefore, DNA fragmentation occurs likely through an oxidative pathway. MTT assays indicated that the complex is highly cytotoxic against three distinct cell lines: B16–F10 (IC₅₀ = 3.39 ± 0.61 μmol L⁻¹), HUH-7 (IC₅₀ = 4.22 ± 0.31 μmol L⁻¹) and 786-0 (IC₅₀ = 10.38 ± 0.91 μmol L⁻¹). Flow cytometry studies conducted with 786-0 cell line indicated cell death might occur by apoptosis. Cell cycle progression evaluated at 5 and 10 μmol L⁻¹ resulted in a clear increase of 786-0 cells at G1 phase and depletion of G2/M, while higher doses showed an expressive increase of sub-G1 phase. Altogether, these results pointed out to a promising biological activity and potential as an anti-cancer agent.

Proposed catalytic cycle for ROS production in the vicinity of DNA after reduction of [Cu(phen)(van)₂] by glutathion.     

A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels,Ca2+ Signaling,and Gene Expression in T Lymphocytes

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K⁺ channels (K_V and K_Ca, respectively), resulting in depolarization of the membrane potential. As a result, Ca²⁺ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca²⁺ signals after thapsigargin stimulation, as well as oscillatory Ca²⁺ signals, but not the Ca²⁺ transient after TCR stimulation. K⁺ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked K_V and K_Ca channels. Progesterone effectively blocked a broad spectrum of K⁺ channels, reducing both Kv1.3 and charybdotoxin–resistant components of K_V current and K_Ca current in T cells, as well as blocking several cloned K_V channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na⁺ channel, an inward rectifier K⁺ channel, or on lymphocyte Ca²⁺ and Cl⁻ channels. We propose that direct inhibition of K⁺ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.     

Design,synthesis, theoretical study,antioxidant, and anticholinesterase activities of new pyrazolo-fused phenanthrolines

Pyrazole-fused phenanthroline compounds were obtained through several synthetic routes. NMR, HRMS, and IR techniques were used to characterize and confirm the chemical structures. Crystal structures were obtained from compounds 3a, 5b, 5j, 5k, and 5n and analyzed using X-ray diffraction. Compounds were evaluated as acetyl (AChE) and butyrylcholinesterase (BChE) inhibitors, and the results showed a moderate activity. Compound 5c presented the best activity against AChE (IC₅₀ = 53.29 μM) and compound 5l against BChE enzyme (IC₅₀ = 119.3 μM). Furthermore, the ability of the synthetic compounds to scavenge cationic radicals DPPH and ABTS was evaluated. Compound 5e (EC₅₀ = 26.71 μg mL⁻¹) presented the best results in the DPPH assay, and compounds 5e, 5f and 5g (EC₅₀ = 11.51, 3.10 and <3 μg mL⁻¹, respectively) showed better ABTS cationic radical scavenging results. Finally, in silico analyses indicated that 71% of the compounds show good oral availability and are within the ranges established by the Lipinski criteria.

Different synthetic strategies were used to optimize, obtaining a series of compounds derived from the pyrazole-fused phenanthroline system.     

In vitro activities of new ketolide, telithromycin, and eight other macrolide antibiotics against Streptococcus pneumoniae having mefA and ermB genes that mediate macrolide resistance

The comparative in vitro activity of a new ketolide, telithromycin (TEL), and eight other macrolide-lincosamide antibiotics (MLS) against 215 strains, of Streptococcus pneumoniae including penicillin-resistant isolates (PRSP), was determined by the agar dilution method. These strains were isolated from patients with pneumonia, otitis media, and purulent meningitis between 1995 and 1997. Two genes, mefA and ermB, that encode MLS resistance in the strains were identified by polymerase chain reaction (PCR). Of the strains, 30.2% (n = 65) had the mefA gene, 37.7% (n = 81) had the ermB gene, and 1.4% (n = 3) had both resistant genes. The minimum inhibitory concentration (MIC_90s) of TEL and 16-membered ring MLS for strains having the mefA gene were 0.063–0.25µg/ml, which were the same level as those for MLS-susceptible strains. On the other hand, the strains with the mefA gene showed low-level resistance to 14- and 15-membered ring MLS, with MIC_90s ranging from 1 to 4µg/ml. Only the MIC₉₀ of TEL at 0.5µg/ml, for strains having the ermB gene was superior to that of the 14-, 15-, and 16-membered ring MLS (MIC₉₀, 64µg/ml). TEL also showed excellent activity against PRSP having abnormal pbp1a, pbp2x, and pbp2b genes. Most strains having the mefA and ermB genes were serotyped to 3, 6, 14, 19, and 23. These results suggest that TEL may be a useful chemotherapeutic agent for respiratory tract infections caused by S. pneumoniae.     

Anti-Trichomonas vaginalis activity of synthetic lipophilic diamine and amino alcohol derivatives

Taking in account the increased prevalence of metronidazole-resistant infections, alternative drugs are necessary for the treatment of trichomonosis. We report in this work the preparation and the in vitro anti-trichomonads activity of two diamines 1 and 2, and three different lipophilic amino alcohol derivatives 3, 4 and 5. These compounds were tested for in vitro activity against two isolates of Trichomonas vaginalis and displayed inhibition of the parasite growth. Five concentrations of each compound were tested. The butanediamine derivative 1, at a final concentration of 5.85 μM, presented a cytotoxic effect against 47% of T. vaginalis trophozoites. Furthermore, the cytotoxicity of 1 did not present statistically significant difference when compared to metronidazole in the same range of concentration (0.1–1.5 μg/mL).     

Biological activity evaluation and action mechanism of chalcone derivatives containing thiophene sulfonate

A series of novel chalcone derivatives containing a thiophene sulfonate group were designed and synthesized. The structures of all title compounds were determined by ¹H-NMR, ¹³C-NMR and HRMS. Antibacterial bioassays indicated that, compound 2l demonstrated excellent antibacterial activities against Xanthomonas axonopodis pv. citri (Xac), with an EC₅₀ value of 11.4 μg mL⁻¹, which is significantly superior to those of bismerthiazol (BT) (51.6 μg mL⁻¹) and thiodiazole-copper (TC) (94.7 μg mL⁻¹). Meanwhile, the mechanism of action of compound 2l was confirmed by using scanning electron microscopy (SEM). In addition, compound 2e showed remarkable inactivation activity against Tobacco mosaic virus (TMV), with an EC₅₀ value of 44.3 μg mL⁻¹, which was superior to that of ningnanmycin (120.6 μg mL⁻¹). Microscale thermophoresis (MST) also showed that the binding of compounds 2e and 2h to Tobacco mosaic virus coat protein (TMV-CP) yielded K_d values of 0.270 and 0.301 μmol L⁻¹, which are better than that of ningnanmycin (0.596 μmol L⁻¹). At the same time, molecular docking studies for 2e and 2h with TMV-CP (PDB code: 1EI7) showed that the compound was embedded well in the pocket between the two subunits of TMV-CP in each case. These results suggested that chalcone derivatives containing a thiophene sulfonate group may be considered as activators in the design of antibacterial and antiviral agents.

Synthesis, antibacterial, antiviral activities and action mechanism of chalcone derivatives containing thiophene sulfonate.     

The effect of pulsed ultrasound exposure on the oxygen dissociation curve of human erythrocytes in in vitro conditions

The P₅₀ value, a marker for the behavior of the oxygen dissociation curve (ODC) and values of 2,3-diphosphoglyceric acid (2,3-DPG), blood gases and acid base parameters (pH, PCO₂, actual HCO₃⁻, base excess, PO₂, SO₂) and erythrocyte parameters (the number of erythrocyte, hematocrit and hemoglobin values) that are known to effect P₅₀, were measured before and after ultrasound (US) exposure to examine the effect of pulsed US beams on the ODC of human erythrocytes. Venous blood samples taken from healthy subjects were sonicated at 0°C for 30 min by pulsed US beams with a constant frequency of 5 MHz. Experiments showed that the values of pH, actual HCO₃⁻, standard HCO₃⁻, base excess, 2,3-DPG and P₅₀ were changed considerably by pulsed US. Furthermore, correlation calculations were made to find out the relation between the P₅₀ values and the other parameters. As a result, a significant relation was found between P₅₀ values and the values of pH, actual HCO₃⁻, PO₂, SO₂ and 2,3-DPG. In conclusion, we can express that a highly significant increase occurs in P₅₀ value of venous blood after a pulsed US exposure at a constant frequency of 5 MHz for 30 min under in vitro conditions at 0⁰C. That is hemoglobin becomes suitable for deoxy (tense structure) conformation when blood affinity of O₂ is low. It means that hemoglobin becomes convenient for proton (H⁺) and CO₂ binding. (E-mail: korpinar@istanbul.edu.tr)     

New chalcone derivatives: synthesis,antiviral activity and mechanism of action

In this work, twenty-eight chalcone derivatives containing a purine (sulfur) ether moiety were synthesized and their antiviral activities were evaluated. Biological results showed that compound 5d exhibited outstanding inactive activity against tobacco mosaic virus (TMV) in vivo (EC₅₀ = 65.8 μg mL⁻¹), which is significantly superior to that of ribavirin (EC₅₀ = 154.3 μg mL⁻¹). Transmission electron microscopy indicated that compound 5d can break the integrity of TMV particles. The results of microscale thermophoresis, fluorescence titration and molecular docking showed that compound 5d had stronger combining affinity (K_a = 1.02 ×10⁵ L mol⁻¹, K_d = 13.4 μmol L⁻¹) with TMV coat protein (TMV-CP), which is due to the formation of five hydrogen bonds between compound 5d and the amino-acid residues of TMV-CP. These findings revealed that compound 5d can effectively inhibit the infective ability of TMV. This work provides inspiration and reference for the discovery of new antiviral agents.

The chalcone derivatives containing a purine (sulfur) ether moiety were synthesized. The antiviral mechanism suggested that the antiviral activity of compound 5d may depend on its stronger binding affinity with TMV-CP.     

Design,synthesis, bio-evaluation,and in silico studies of some N-substituted 6-(chloro/nitro)-1H-benzimidazole derivatives as antimicrobial and anticancer agents

A new series of 6-substituted 1H-benzimidazole derivatives were synthesized by reacting various substituted aromatic aldehydes with 4-nitro-o-phenylenediamine and 4-chloro-o-phenylenediamine through condensation using sodium metabisulfite as the oxidative reagent. The N-substituted 6-(chloro/nitro)-1H-benzimidazole derivatives were prepared from the 6-substituted 1H-benzimidazole derivatives and substituted halides using potassium carbonate by conventional methods as well as by exposure to microwave irradiation. Seventy-six 1H-benzimidazole derivatives have been synthesized in moderate to excellent yields with the microwave-assisted method (40 to 99%). Compounds 1d, 2d, 3s, 4b, and 4k showed potent antibacterial activity against Escherichia coli, Streptococcus faecalis, MSSA (methicillin-susceptible strains of Staphylococcus aureus), and MRSA (methicillin-resistant strains of Staphylococcus aureus) with MIC (the minimum inhibitory concentration) ranging between 2 and 16 μg mL⁻¹ as compared to ciprofloxacin (MIC = 8–16 μg mL⁻¹), in particular compound 4k exhibits potent fungal activity against Candida albicans and Aspergillus niger with MIC ranging between 8 and 16 μg mL⁻¹ compared with the standard drug fluconazole (MIC = 4–128 μg mL⁻¹). In addition, compounds 1d, 2d, 3s, 4b, and 4k also showed the strongest anticancer activity among the synthesized compounds against five tested cell lines with IC₅₀ (half-maximal inhibitory concentration) ranging between 1.84 and 10.28 μg mL⁻¹, comparable to paclitaxel (IC₅₀ = 1.38–6.13 μM). Furthermore, the five most active compounds showed a good ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile in comparison to ciprofloxacin, fluconazole, and paclitaxel as reference drugs. Molecular docking predicted that dihydrofolate reductase protein from Staphylococcus aureus is the most suitable target for both antimicrobial and anticancer activities, and vascular endothelial growth factor receptor 2 and histone deacetylase 6 are the most suitable targets for anticancer activity of these potent compounds.

The purpose of this study is to synthesize novel N-substituted 6-(chloro/nitro)-1H-benzimidazole derivatives with various substituted aryl groups at position 2 and alkylation at position 1, and evaluate their antimicrobial and anticancer activities.     

Thyrotropin-releasing Hormone Stimulation of Prolactin Release from Clonal Rat Pituitary Cells: EVIDENCE FOR ACTION INDEPENDENT OF EXTRACELLULAR CALCIUM

Thyrotropin-releasing hormone (TRH) stimulates prolactin release and ⁴⁵Ca²⁺ efflux from GH₃ cells, a clonal strain of rat pituitary cells. Elevation of extracellular K⁺ also induces prolactin release and increases ⁴⁵Ca²⁺ efflux from these cells. In this report, we distinguish between TRH and high K⁺ as secretagogues and show that TRH-induced release of prolactin and ⁴⁵Ca²⁺ is independent of the extracellular Ca²⁺ concentration, but the effect of high K⁺ on prolactin release and ⁴⁵Ca²⁺ efflux is dependent on the concentration of Ca²⁺ in the medium. The increment in ⁴⁵Ca²⁺ efflux induced by 50 mM K⁺ during perifusion was reduced in a concentration-dependent manner by lowering extracellular Ca²⁺ from 1,500 to 0.02 μM (by adding EGTA), whereas 1 μM TRH enhanced ⁴⁵Ca²⁺ efflux similarly over the entire range of extracellular Ca²⁺ concentrations. Although 50 mM K⁺ caused release of 150 ng prolactin from 40 × 10⁶ GH₃ cells exposed to 1,500 μM Ca²⁺ (control), reduction of extracellular Ca²⁺ to 2.8 μM decreased prolactin release caused by high K⁺ to <3% of controls and no prolactin release was detected after exposure to 50 mM K⁺ in medium with 0.02 μM free Ca²⁺. In contrast, TRH caused release of 64 ng of prolactin from 40 × 10⁶ GH₃ cells exposed to medium with 1,500 μM Ca²⁺, and release caused by TRH was still 50 and 35% of control in medium with 2.8 and 0.02 μM Ca²⁺, respectively. Furthermore, TRH transiently increased by 10-fold the fractional efflux of ⁴⁵Ca²⁺ from GH₃ cells in static incubations with 1,500 or 3.5 μM Ca²⁺, hereby confirming that the enhanced ⁴⁵Ca²⁺ efflux caused by TRH in both low and high Ca²⁺ medium was not an artifact of the perifusion system.     

Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis

Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR^® Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host–parasite relationship.     

Synthesis, cytotoxicity, and in vitro antileishmanial activity of mono-t-butyloxycarbonyl-protected diamines

Leishmania amazonensis is the etiologic agent of the cutaneous and diffuse leishmaniasis. This species is often associated with drug resistance, and the conventional treatments exhibit high toxicity for patients. Therefore, the search for new antileishmanial compounds is urgently needed since there is no vaccine available. In this study, using the in vitro traditional drug screening test, we have analyzed the effects of a series of diaminoalkanes monoprotected with t-butyloxycarbonyl (BOC) against L. amazonensis. Among the 18 tested compounds, 6 exhibited antileishmanial activity (2, 7–9, 17, and 18). Best IC₅₀ values (10.39 ± 0.27 and 3.8 ± 0.42 μg/mL) were observed for compounds 17 and 18 (H₂N(CH₂)nNHBoc, n = 10 and 12), respectively. Although those compounds had higher lipophilicity as indicated by their cLog P values, compound 17 was very toxic. Determination of the selective indexes indicated that 50% of the active compounds were very toxic for HepG2 cells. However, compounds 2, 8, and 18 had good lipophilicity and were less toxic among all polyamine derivatives tested. The chemical properties of antileishmanial diamine derivatives, such as lipophilicity and cytotoxicity, are relevant factors for the design of new drugs. A higher lipophilicity is likely to improve the chances of reaching this intracellular parasite.