Effects of N-acetylcystein on bleomycin-induced apoptosis in malignant testicular germ cell tumors

Oxidative stress has been shown to induce apoptosis in cancer cells. Therefore, one might suspect that antioxidants may inhibit reactive oxygen species (ROS) and prevent apoptosis of cancer cells. No study has been carried out so far to elucidate the effects of N-acetylcysteine (NAC) on bleomycin-induced apoptosis in human testicular cancer (NCCIT) cells. We investigated the molecular mechanisms of apoptosis induced by bleomycin and the effect of NAC in NCCIT cells. We compared the effects of bleomycin on apoptosis with H₂O₂ which directly produces ROS. Strong antioxidant NAC was evaluated alone and in combination with bleomycin or H₂O₂ in germ cell tumor-derived NCCIT cell line (embryonal carcinoma, being the nonseminomatous stem cell component). We determined the cytotoxic effect of bleomycin and H₂O₂ on NCCIT cells and measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and cytochrome c (Cyt-c) levels in NCCIT cells incubated with bleomycin, H₂O₂, and/or NAC. We found half of the lethal dose (LD₅₀) of bleomycin on NCCIT cell viability as 120???g/ml after incubation for 72?h. Incubation with bleomycin (LD₅₀) induced increases in caspase-3, caspase-8, and caspase-9 activities and Cyt-c and Bax protein levels and a decrease in Bcl-2 level. Co-incubation of NCCIT cells with bleomycin and 10?mM NAC abolished bleomycin-induced increases in caspase-3 and caspase-9 activities, Bax, and Cyt-c levels and bleomycin-induced decrease in Bcl-2 level. Our results indicate that bleomycin induces apoptosis in NICCT cells and that NAC diminishes bleomycin-induced apoptosis via inhibiting the mitochondrial pathway. We conclude that NAC has negative effects on bleomycin-induced apoptosis in NICCT cells and causes resistance to apoptosis, which is not a desirable effect in the fight against cancer.     

Synthesis and biological activity of some new flavonyl-2,4-thiazolidinediones

A new series of flavonyl-2,4-thiazolidinediones (Va-c, VIa-c) was prepared by Knoevenagel reaction. The synthesized compounds were tested for their ability to inhibit rat kidney aldose reductase (AR) and for their insulinotropic activities in INS-1 cells. Compound Vb was able to increase insulin release in the presence of 5.6mmol/l glucose. Compounds VIa-c displayed moderate to high AR inhibitory activity levels. Particularly, compound VIa showed the highest AR inhibitory activity (86.57%).     

In vitro aldose reductase inhibitory activity of some flavonyl-2,4-thiazolidinediones

Aldose reductase (AR) is implicated to play a critical role in diabetes and cardiovascular complications because of the reaction it catalyzes. AR enzyme appears to be the key factor in the reduction of glucose to sorbitol. Synthesis and accumulation of sorbitol in cells due to AR activity is the main cause of diabetic complications, such as diabetic cataract, retinopathy, neuropathy and nephropathy. Aldose reductase inhibitors have been found to prevent sorbitol accumulation in tissues. Numerous compounds have been prepared in order to improve the pharmacological prophile of inhibition of aldose reductase enzyme. In this study, seventeen flavonyl-2,4-thiazolidinediones (flavonyl-2,4-TZD) (Ia-e, IIa-e and IIIa-g) were tested for their ability to inhibit rat kidney AR. Compound Ib showed the highest inhibitory activity (88.69 +/- 1.46%) whereas Ia, IIa, IIIa, IIIb also showed significant inhibitory activity (49.26 +/- 2.85, 67.29 +/- 1.09, 71.11 +/- 1.95, 64.86 +/- 1.21%, respectively).     

Some benzoxazoles and benzimidazoles as DNA topoisomerase I and II inhibitors

Some novel fused heterocyclic compounds of 2, 5-disubstituted-benzoxazole and benzimidazole derivatives, which were previously synthesized by our group, were investigated for their inhibitory activity on both eukaryotic DNA topoisomerase I and II in a cell free system. 2-Phenoxymethylbenzimidazole (17), 5-amino-2-(p-fluorophenyl)benzoxazole (3), 5-amino-2-(p-bromophenyl)benzoxazole (5), 5-nitro-2-phenoxymethyl-benzimidazole (18), 2-(p-chlorobenzyl)benzoxazole (10) and 5-amino-2-phenylbenzoxazole (2) were found to be more potent as eukaryotic DNA topoisomerase I poisons than the reference drug camptothecin having IC(50) values of 14.1, 132.3, 134.1, 248, 443.5, and 495 microM, respectively. 5-Chloro-2-(p-methylphenyl)benzoxazole (4), 2-(p-nitrobenzyl)benzoxazole (6) and 5-nitro-2-(p-nitrobenzyl)benzoxazole (8) exhibited significant activity as eukaryotic DNA topoisomerase II inhibitors, having IC(50) values of 22.3, 17.4, 91.41 microM, respectively, showing higher potency than the reference drug etoposide.     

Comparison of some properties of free and immobilized alpha-amylase by Aspergillus sclerotiorum in calcium alginate gel beads

Some properties of immobilized alpha-amylase by Aspergillus sclerotiorum within calcium alginate gel beads were investigated and compared with soluble enzyme. Optimum pH and temperature were found to be 5.0 and 40 degrees C, respectively, for both soluble and immobilized enzymes. The immobilized enzyme had a better Km value, but kcat/Km values were the same for both enzymes. Entrapment within calcium alginate gel beads improved, remarkably, the thermal and storage stability of alpha-amylase. The half life values of immobilized enzyme and soluble enzyme at 60 degrees C were 164.2, and 26.2 min, respectively. The midpoint of thermal inactivation (Tm) shifted from 56 degrees C (for soluble enzyme) to 65.4 degrees C for immobilized enzyme. The percentages of soluble starch hydrolysis for soluble and immobilized alpha-amylase were determined to be 97.5 and 92.2% for 60 min, respectively.     

Synthesis and pharmacology of the enantiomers of UH301: Opposing interactions with 5-HT1A receptors

The (S)-enantiomer of 5-fluoro-8-hydroxy-2-(dipropylamino) tetralin [(S)- 2a; (S)-UH301] was the first reported 5-HT_1A receptor antagonist. We now give a full account on the synthetic effort leading to the preparation of the racemate and the enantiomers of 2a. The crystal and molecular structure of 2a · HBr has been determined by X-ray diffraction and the absolute configuration has been deduced using statistical tests of the crystallographic R values. The unit cell is tetragonal (P4₁2₁2) with a = b = 13.2235 (2), c = 39.560(1) Å and contains two crystallographically independent molecules in each asymmetric unit. The two solid state conformers differ in the conformation of the N-propyl groups. The pharmacological characterization of the enantiomers was done by use of in vivo biochemical and behavioural assays in rats. The (R)-enantiomer of 2a is a 5-HT_1A receptor agonist of low potency while (S)- 2a does not exhibit any agonist properties at 5-HT_1A receptors. As a consequence of the opposing effects of the enantiomers, the racemate, rac- 2a, does not produce any clear-cut effects in rats. The reduced efficacy of (S)- 2a as compared to the well known 5-HT_1A receptor agonist 8-hydroxy-2-(dipropylamino)tetralin ( 1; 8-OH-DPAT) may be due to the fluoro-substituent induced negative potential of the aromatic ring. Chirality 8:531–544, 1996. © 1997 Wiley-Liss, Inc.     

Serum selenium and gluthathione-peroxidase activities and their interaction with toxic metals in dialysis and renal transplantation patients

Selenium, aluminum, cadmium, and magnesium concentrations and gluthathione-peroxidase activities in sera of 35 healthy individuals, 30 renal transplants, and 30 hemodialysis patients were measured. Serum selenium, aluminum, and cadmium concentrations in both groups of patients were higher than the controls (p<0.001), whereas the serum gluthathione-peroxidase levels were lower (p<0.001). According to our results, it can be concluded that the patients receiving hemodialysis are subjected to more toxic elements than the transplantation patients. These findings imply that dietary selenium supplement may be suggested in renal failure for the detoxification of elements, such as cadmium and mercury. The essential trace element selenium takes part not only in the direct protection of endothelial cells against the accumulation of aggressive oxygen species, but also in the preventions of the toxic effects of cadmium or in the modulation of the active calcium transport.     

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent K _m values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent K _m values were 0.1 mM for -ketoglutarate and 0.22 mM for glutamine.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations.

Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.