Cytotoxicity of boron containing dipeptide analogs

Summary This work is an extension of our previous work (Hall et al., 1993) on the synthesis and cytotoxic activity of boronated peptides. The aim of this work was to carry out structural modifications of the amine terminal in compounds1 and2, to increase water solubility, and its effect on the cytotoxicity to tumor cell lines. Surprisingly, only compounds4,7 and8 were more water soluble than the parent compounds. With the exception of compound4, the new derivatives were generally less effective than the parent compounds (1 and2). There was no apparent correlation between structure and activity. Cytotoxic effect was more pronounced in single cell suspended cells. The growth of solid tumor cell lines was not significantly reduced. The most active derivative, (methanamine)dihydro[[[1-(phenylmethyl)-2-methylamino-2-oxoethyl]amino]carboxy]boron (4), inhibited DNA, RNA, and protein synthesis in Tmolt₃ cells. Enzymatic activities, e.g., DNA polymerase, m-RNA polymerase, PRPP amidotransferase, carbamyl phosphate synthetase, TMP-kinase, TDP-kinase, dihydrofolate reductase, and ribonucleoside reductase were reduced after 60 min incubation with4. d(TTP) and d(CTP) pool levels were also reduced by 60 min incubation with4.     

Synthesis and preliminary screening of novel N-{2-[4-(substituted)piperazin-1-yl]-2-oxoethyl}acetamides as potential atypical antipsychotic agents

A series of N-{2-[4-(substituted)piperazin-1-yl]-2-oxoethyl}acetamides were synthesized as prospective novel atypical antipsychotic agents. Microwave irradiation of acetyl glycine (I) with substituted piperazines in the presence of DCC in DMF for about 3-5 min gave the titled compounds (P:1-7). All the synthesized compounds were screened for their in vivo pharmacological activity in Swiss albino mice. D₂ antagonism studies were performed using the climbing mouse assay model and 5-HT_2A antagonism studies were performed using quipazine induced head twitches in mice. Among the synthesized compounds P4 was found to be the most active compound.     

In vivo and in vitro effect of androstene derivatives as 5α-reductase type 1 enzyme inhibitors

The aim of these studies was to synthesize twelve ester derivatives of dehydroepiandrosterone with therapeutic potential. The effect of 1–12 was demonstrated in the flank organs of gonadectomized hamsters treated with testosterone and the synthesized steroids. In vitro studies were carried out determining the IC₅₀ values for the inhibition of the activity of 5α-reductase type 1 and 2, which are present in rat liver and human prostate respectively. The binding of 1–12 to the androgen receptors (AR) was determined using rat’s prostate cytosol. Steroids 1–12 containing different substituents in the phenyl group of the ester moiety in C-3 reduced the flank organs and inhibited the activity of 5α-R type 1; however only steroids 1 and 2 inhibited 5α-R type 2. 1–12 did not bind to the AR. The modification of one atom of the substituents in the phenyl group of the ester moiety in C-3 changed their biological potency (IC₅₀).     

Apical and basolateral Na/H exchange in cultured murine proximal tubule cells (MCT): Effect of parathyroid hormone (PTH)

Summary Kidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10^–8 m; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10^–8 m); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10^–8 m) resulted in a rapid and transient increase in [Ca²⁺] _i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca²⁺ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/P _i cotransport.This work was supported by the Swiss National Science Foundation (Grant No. 32-30785.91), the Stiftung für wissenschaftliche Forschung an der Universität Zürich, the Hartmann-Müller Stiftung, the Sandoz-Stiftung, the Roche Research Foundation and the Geigy-Jubiläumsstiftung. We are grateful to Denise Rossi and Christa Knellwolf for their excellent secretarial assistance.     

Podophyllum hexandrum aqueous extract as a potential free radical scavenger

Abstract

The present study was undertaken to evaluate the effect of the aqueous extract of Podophyllum hexandrum against free radical-mediated damage and also explore its anticancer activity. The extract exhibited significant activity in scavenging 1, 1-diphenyl-2-picryl-hydrazyl radicals, ^?OH radical-mediated DNA damage, and lipid peroxide production in rat liver microsomes. The extract was also tested for its reducing abilities. The activity of liver marker enzymes and antioxidant defense enzymes in rat liver homogenate was assessed in control and carbon tetrachloride (CCl₄)-treated animals. It was observed that CCl₄-induced changes viz., increases in the activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, a decrease in reduced glutathione as well as decreases in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. All these parameters showed reversal when pretreated with aqueous extract of P. hexandrum. Podophylotoxin and etoposide are the two known anticancer agents derived from P. hexandrum and interestingly the aqueous extract of P. hexandrum showed a typical DNA ladder formation in HL-60 cells confirming its role as an inducer of apoptosis. The results obtained suggest that the plant extract exhibits inhibition of and free radical production and lipid peroxidation, increase in antioxidant enzyme activities, revealing its antioxidant properties, and is also able to show potent anticancer activity as depicted by its ability to cause fragmentation of DNA.     

Bioorganometallics: First examples of cyclometalated iridium(III) complexes containing di- and tripeptide ester ligands

The synthesis of bis-cyclometalated [Ir(ptpy)₂(gly-gly-OEt)] (2, ptpy = 2-(p-tolyl)pyridinato; gly-gly-OEt = glycylglycine ethyl ester) and [Ir(ptpy)₂(gly-gly-gly-OEt)] (3, gly-gly-gly-OEt = glycylglycylglycine ethyl ester) from the reaction of [{Ir(μ-Cl)(ptpy)₂}₂] (1) with the corresponding peptide ester hydrochlorides in the presence of NaOMe is described. The molecular structure of 2 was confirmed by a single-crystal X-ray diffraction study. The compound crystallized from dichloromethane/iso-hexane in the space group P2₁/a. In the crystal packing the molecules of 2 exhibit N–H?O hydrogen bonds to the neighbor molecules to form dimeric units. The absorption and emission spectra of 2 and 3 were recorded and exhibit these compounds as strong green-emitting complexes.     

A comparative study on the antimutagenicity of atorvastatin and lovastatin against directly acting mutagens

Elevated levels of oxidative DNA lesions have been noted in many tumors and such damage is strongly implicated in the etiology of cancer. The cumulative risk of cancer increases with the fourth power of age and is associated with an accumulation of oxidative DNA damage. Many agents, synthetic or natural, that can inhibit mutation have been depicted as cancer chemopreventive agents. Antimutagenicity of the 3-hydroxy-3-methylgutaryl-CoA (HMG-CoA) reductase inhibitors atorvastatin and lovastatin was studied using the Ames Salmonella typhimurium assay. Directly acting mutagens, sodium azide (NaN₃) and 4-nitro-o-phenylenediamine (NPDA), were used to induce mutation in Salmonella strains TA98 and TA100. The antimutagenicity of lovastatin and atorvastatin was found to be significant (p < 0.01) and dose-dependent. The percentage inhibition of a 3 mg lovastatin-treated plate was found to be 79.9% and 61.8% against NPDA- and NaN₃-induced mutation to TA98 and TA100, respectively. Atorvastatin (0.5 mg/plate) inhibited NPDA-and NaN₃-induced mutation to TA98 and TA100 by 78.6% and 45.5%, respectively. Atorvastatin showed antimutagenic activity at lower concentrations than lovatstatin. The results of the present study regarding the antimutagenic activity of atorvastatin and lovastatin suggested their therapeutic application as cancer chemopreventive agents.     

Unusual stability exhibited by (AT)XN12(AT)Y motif associated with high fetal hemoglobin levels

Abstract

Quasi-palindromic sequences (AT)_XN₁₂(AT)_Y present in HS2 (hypersensitive site 2) of the human β-globin locus are known to be significantly associated with increased fetal hemoglobin (HbF) levels. High HbF levels in some adults arise due to pathological conditions such as sickle cell disease and β-thalassemia. However, elevated levels of HbF are also associated with a reducing morbidity and mortality in patients with β-thalassemia and thus ameliorate the severity of the disease. Using gel-electrophoresis, ultraviolet (UV)-thermal denaturation, and circular dichroism (CD) techniques, we demonstrated that it exhibits a hairpin-duplex equilibrium. Intramolecular species (hairpin) were observed in both low and high salt concentrations in gel assay studies displaying the unusual stability of intramolecular species even at the high counter-ion concentration. The unusual stability of hairpin secondary structures was also demonstrated by the monophasic nature of the melting profiles for the oligonucleotides which persisted at low as well as high salt and oligomer concentrations. Change in CD spectra as a function of oligomer concentration indicates that the bimolecular duplex formation is selectively favored over monomolecular hairpin formation at and above 9 µM oligomer concentration. Thus, we hypothesize that imperfect inverted repeat sequence (AT)_XN₁₂(AT)_Y of HS2 of β-globin gene LCR forms the unusually stable hairpins which may result in the formation of a cruciform structure that may be recruited for binding by various nuclear proteins that could result in elevated HbF levels.

Communicated by Ramaswamy H. Sarma.     

Antitumor effects of a novel benzonaphthofurandione derivative (8e) on the human colon cancer cells in vitro and in vivo through cell cycle arrest accompanied with the modulation of EGFR and mTOR signaling

Benzonaphthofurandione has been considered as an important class of naturally occurring and synthetic compounds having a variety of biological functions. In this study, we evaluated the antitumor effects of 3-[2-(dimethylamino)isopropoxy]-1-hydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione (8e), a novel benzonaphthofurandione derivative, on the growth of colorectal cancer HCT 116 cells both in vitro culture and an in vivo animal model.Compound 8e exhibited the potential growth inhibition of the colon cancer cells in a concentration-dependent manner. The anti-proliferative activity of 8e was also associated with the induction of cell cycle arrest in the G₀/G₁ phase. The 8e-induced cell cycle arrest was well correlated with the suppression of cyclin-dependent kinase 2 (CDK2), CDK4, cyclin D1, cyclin E, c-Myc, and phosphorylated retinoblastoma protein (pRb). The tumor growth in xenograft nude mice bearing HCT 116 cells by compound 8e (10 mg/kg) also significantly inhibited without any overt toxicity. In addition, the down-regulation of epidermal growth factor receptor (EGFR), Akt, and mTOR signalings were associated with the anti-proliferative activity of compound 8e in colon cancer cells. Taken together, these findings suggested that cell cycle arrest and modulation of cell signal transduction pathways might be the plausible mechanisms of actions for the anti-proliferative activity of 8e, and thus 8e might be used as an effective chemotherapeutic agent in human colon cancer.     

Water transport and estimated transmembrane potential during freezing of mouse oocytes

Summary Steady-state currents at hyperpolarized membrane potentials were studied in the homologous giant neurons, LP1 and R2, ofAplysia using two-electrode voltage clamp. Nearly half of the steady-state current at voltages more hyperpolarized than –70 mV had characteristics similar to the inwardly rectifying potassium current (I _R) described previously inAplysia neurons. The pharmacological agents 4-bromophenacylbromide, indomethacin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate were found to modulateI _R.I _R was stimulated with BPB and indomethacin and inhibited with TPA. These agents alteredI _R by a mechanism independent ofcAMP, which can also modulateI _R. The effects of these modulators are consistent with their actions on arachidonic acid (AA) metabolism inAplysia nervous system, suggesting AA may constitutively inhibitI _R. When ganglia were perfused for 12 hr with medium containing BSA to absorb extracellular fatty acids,I _R was increased nearly twofold. This increase was partially inhibited by addition of AA to the perfusion medium, and completely inhibited by pretreatment of ganglia with BPB. Although no direct effect of shortterm exposure to exogenous AA was observed, long term exposure to exogenous AA and several other unsaturated fatty acids was accompanied by a decrease inI _R.     

Syntheses and characterization of vitamin B<Subscript>12</Subscript>–Pt(II) conjugates and their adenosylation in an enzymatic assay

Aiming at the use of vitamin B₁₂ as a drug delivery carrier for cytotoxic agents, we have reacted vitamin B₁₂ with trans-[PtCl(NH₃)₂(H₂O)]⁺, [PtCl₃(NH₃)]⁻ and [PtCl₄]²⁻. These Pt(II) precursors coordinated directly to the Co(III)-bound cyanide, giving the conjugates [{Co}–CN–{trans-PtCl(NH₃)₂}]⁺ (5), [{Co}–CN–{trans-PtCl₂(NH₃)}] (6), [{Co}–CN–{cis-PtCl₂(NH₃)}] (7) and [{Co}–CN–{PtCl₃}]⁻ (8) in good yields. Spectroscopic analyses for all compounds and X-ray structure elucidation for 5 and 7 confirmed their authenticity and the presence of the central “Co–CN–Pt” motif. Applicability of these heterodinuclear conjugates depends primarily on serum stability. Whereas 6 and 8 transmetallated rapidly to bovine serum albumin proteins, compounds 5 and 7 were reasonably stable. Around 20% of cyanocobalamin could be detected after 48 h, while the remaining 80% was still the respective vitamin B₁₂ conjugates. Release of the platinum complexes from vitamin B₁₂ is driven by intracellular reduction of Co(III) to Co(II) to Co(I) and subsequent adenosylation by the adenosyltransferase CobA. Despite bearing a rather large metal complex on the β-axial position, the cobamides in 5 and 7 are recognized by the corrinoid adenosyltransferase enzyme that catalyzes the formation of the organometallic C–Co bond present in adenosylcobalamin after release of the Pt(II) complexes. Thus, vitamin B₁₂ can potentially be used for delivering metal-containing compounds into cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Antiplatelet pyrazolopyridines derivatives: pharmacological,biochemical and toxicological characterization

Platelet aggregation is one of the main events involved in vascular thrombus formation. Recently, N′-substituted-phenylmethylene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazides were described as antiplatelet derivatives. In this work, we explore the properties of these antiplatelet agents through a series of pharmacological, biochemical and toxicological studies. The antiplatelet activity of each derivative was confirmed as 3a, 3b and 3?h significantly inhibited human platelet aggregation induced by arachidonic acid, with no detectable effect on clotting factors or healthy erythrocytes. Importantly, mice treated with derivative 3a showed a higher survival rate at an in vivo model of pulmonary thromboembolism with a lower bleeding risk in comparison to aspirin. The in silico studies pointed a series of structural parameters related to thromboxane synthase (TXS) inhibition by 3a, which was confirmed by tracking plasma levels of PGE₂ and TXB₂ through an in vitro enzyme immunoassay. Derivative 3a showed selective TXS inhibition allied with low bleeding risk and increased animal survival, revealing the derivative as a promising candidate for treatment of cardiovascular diseases.     

Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

Titanium dioxide nanoparticles preferentially bind in subdomains IB,IIA of HSA and minor groove of DNA

Titanium dioxide nanoparticles (TiO₂-NPs) interaction with human serum albumin (HSA) and DNA was studied by UV–visible spectroscopy, spectrofluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) to analyze the binding parameters and protein corona formation. TEM revealed protein corona formation on TiO₂-NPs surface due to adsorption of HSA. Intrinsic fluorescence quenching data suggested significant binding of TiO₂-NPs (avg. size 14.0 nm) with HSA. The Stern–Volmer constant (K_sv) was determined to be 7.6 × 10² M^?1 (r² = 0.98), whereas the binding constant (K_a) and number of binding sites (n) were assessed to be 5.82 × 10² M^?1 and 0.97, respectively. Synchronous fluorescence revealed an apparent decrease in fluorescence intensity with a red shift of 2 nm at Δλ = 15 nm and Δλ = 60 nm. UV–visible analysis also provided the binding constant values for TiO₂-NPs–HSA and TiO₂-NPs-DNA complexes as 2.8 × 10² M^?1 and 5.4 × 10³ M^?1. The CD data demonstrated loss in α-helicity of HSA and transformation into β-sheet, suggesting structural alterations by TiO₂-NPs. The docking analysis of TiO₂-NPs with HSA revealed its preferential binding with aromatic and non-aromatic amino acids in subdomain IIA and IB hydrophobic cavity of HSA. Also, the TiO₂-NPs docking revealed the selective binding with A-T bases in minor groove of DNA.     

The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2-isoprostanes) are not correlated in healthy human subjects

Oxidative damage to DNA in human tissues can be determined by measuring multiple products of oxidative damage to the purine and pyrimidine bases using gas chromatography-mass spectrometry (GC-MS). Oxidative damage to lipids (lipid peroxidation) can be quantitated by the mass spectrometry-based determination of F₂-isoprostanes, specific end-products of the peroxidation of arachidonic acid residues in lipids. For both DNA base damage products and 8-epi prostaglandin F_2α (PGF_2α), there is a wide variation in levels between different healthy human subjects. We measured multiple products of oxidative damage to DNA bases in white cells, and 8-epi PGF_2α in plasma, from blood samples obtained from healthy human subjects in the UK and in Portugal. No correlation of 8-epi PGF_2α levels with levels of any modified DNA base (including 8-hydroxyguanine) was observed. We conclude that no single parameter can be measured as an index of “oxidative stress” or “oxidative damage” in vivo.     

WQ-3810 exerts high inhibitory effect on quinolone-resistant DNA gyrase of Salmonella Typhimurium

ABSTRACT

The inhibitory effect of WQ-3810 on DNA gyrase was assayed to evaluate the potential of WQ-3810 as a candidate drug for the treatment of quinolone resistant Salmonella Typhymurium infection. The inhibitory effect of WQ-3810, ciprofloxacin and nalidixic acid was compared by accessing the drug concentration that halves the enzyme activity (IC₅₀) of purified S. Typhimurium wildtype and mutant DNA gyrase with amino acid substitution at position 83 or/and 87 in subunit A (GyrA) causing quinolone resistance. As a result, WQ-3810 reduced the enzyme activity of both wildtype and mutant DNA gyrase at a lower concentration than ciprofloxacin and nalidixic acid. Remarkably, WQ-3810 showed a higher inhibitory effect on DNA gyrase with amino acid substitutions at position 87 than with that at position 83 in GyrA. This study revealed that WQ-3810 could be an effective therapeutic agent, especially against quinolone resistant Salmonella enterica having amino acid substitution at position 87.     

Synthesis and characterisation of (Z)-styrylbenzene derivatives as potential selective anticancer agents

To identify anticancer agents with high potency and low toxicity, a series of (Z)-styrylbenzene derivatives were synthesised and evaluated for anticancer activities using a panel of nine cancer cell lines and two noncancerous cell lines. Most derivatives exhibited significant anti-proliferative activities against five cancer cell lines, including MGC-803 and BEL-7402. (Z)-3-(p-Tolyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (6h) showed a strong inhibitory effect on MGC-803 cells (IC₅₀?50?50 value of 6h in L-02 cells was 10,000-fold higher than in MGC-803 cells. Compound 6h inhibited proliferation of BEL-7402 cells by arresting at the G2/M phase through up-regulation of cyclin B1 expression, down-regulation of cyclin A and D1 expression, and induction of apoptosis. In addition, 6h inhibited the migration of BEL-7402 cells and the formation of cell colonies.     

Ubiquinol-10 and ubiquinone-10 levels in umbilical cord blood of healthy foetuses and the venous blood of their mothers

Abstract

Despite their being good markers of oxidative stress for clinical use, little is known about ubiquinol-10 (reduced coenzyme Q₁₀) and ubiquinone-10 (oxidized coenzyme Q₁₀) levels in foetuses and their mothers. This study investigates oxidative stress in 10 healthy maternal venous, umbilical arterial and venous bloods after vaginal delivery by measuring ubiquinol-10 and ubiquinone-10 levels. Serum ubiquinol-10 and ubiquinone-10 levels were measured by HPLC with a highly sensitive electrochemical detector. Maternal venous ubiquinol-10 and ubiquinone-10 levels were significantly higher than umbilical arterial and venous levels (all p < 0.001). However, the ubiquinone-10/total coenzyme Q₁₀ (CoQ₁₀) ratio, which reflects the redox status, was significantly higher in umbilical arterial and umbilical venous blood compared to maternal venous blood (all p < 0.001). The ubiquinone-10/total CoQ₁₀ ratio was higher in umbilical arterial than in umbilical venous blood (p < 0.01). The present study demonstrated that foetuses were under higher oxidative stress than their mothers.     

Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210

Abstract

Background. Insulin protects cardiomyocytes from reactive oxygen species (ROS)-induced apoptosis after ischemic/reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from ROS injury. Methods. Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H₂O₂) or insulin at various concentrations for various periods of time, or with insulin and H₂O₂ for various periods of time. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results. The percentage of viable cells was significantly and inversely associated with H₂O₂ concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H₂O₂ or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H₂O₂-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion. Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H₂O₂/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.     

Anticholinesterase activity of some major intermediates in carbacylamidophosphate synthesis: Preparation,spectral characterization and inhibitory potency determination

Carbacylamidophosphates with the general formula RC(O)NHP(O)R₁R₂ constitute organophosphorus compounds that are used as insecticides, pesticides and ureas inhibitors. In this work, we studied the inhibition potency of CCl₃C(O)NHP(O)Cl₂1, CHCl₂C(O)NHP(O)Cl₂2, CH₂ClC(O)NHP(O)Cl₂3 and CF₃C(O)NHP(O)Cl₂4, which are the major intermediates for carbacylamidophosphates synthesis towards human erythrocyte acetylcholinesterase (hAChe) activity using Ellman's modified kinetic method. Unexpectedly, it was observed that they were not only hydrolytically unstable but also inhibited hAChE in a similar manner to that produced by organophosphorus insecticides. Enzymatic data, bimolecular inhibition rate constants (k_i) and IC₅₀ values for inhibition of hAChE demonstrated that they are irreversible inhibitors and the inhibition potency of compound 2 (IC₅₀ = 88 μM) was the greatest in comparison with compounds 1, 3 and 4. Also the electropositivity of the phosphorus atom and the hydrophobicity of the compounds demonstrated that these two factors play an additional effect and different role in the inhibitory activity of these compounds. Hydrolytic stability of the compounds was determined by ³¹P NMR monitoring of the loss of the parent molecules with D₂O as a function of time. This study considers antiacetylcholinesterase activity according to the structural and the electronic aspects of compounds 1–4, according to IR, ¹H, ¹³C and ³¹P NMR spectral data.