High-pressure liquid chromatographic analysis for identification of in vitro and in vivo metabolites of 4-phenethyl-5-[4-(1-(2-hydroxyethyl)-3,5-dimethyl-4-pyrazolylazo)phenyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione in rats

All azo colorants whose metabolism can liberate a carcinogenic arylamine, are suspected of having carcinogenic potential. Therefore, a new azo compound 4-phenethyl-5-[4-(1-(2-hydroxyethyl)-3,5-dimethyl-4-pyrazolylazo)phenyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (substrate) was prepared to investigate its in vitro and in vivo biotransformation in rats by HPLC. Chromatographic separation of substrate and its metabolites was performed using a Chromasil C(18) column. The mobile phase consisted of acetonitrile and water in a linear gradient system. From the biotransformation of this compound, the reduction metabolite 4-(2-phenethyl)-5-(4-aminophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione was identified by comparing it to reference standard by HPLC-DAD. In the in vivo study, identification of the unknown peak which was the N-acetylation metabolite was confirmed by LC-MS spectrometry. Besides this, the azo compound was reduced to its corresponding amine in intestinal and cytosolic parts. In addition, oxidation of the methyl group and the phenyl ring, and reduction of azo group to hydrazo were identified in the cytosolic part using LC-MS.     

The first example of calix[4]pyrrole functionalized vic-dioxime ligand: Synthesis, characterization, spectroscopic studies and redox properties of the mononuclear transition metal complexes

Novel calix[4]pyrrole bearing vic-dioxime ligand (LH₂) of the general formula, R₁R₂C₂N₂O₂H₂ (where, R₁ = C₆H₅- and R₂ = C₃₉H₅₀N₅-) has been synthesized by the reaction of anti-chlorophenylglyoxime with 3-aminophenylcalix[4]pyrrole at room temperature. The mononuclear Cu(II), Ni(II) and Co(II)complexes of this vic-dioxime ligand were prepared and their structures were confirmed by elemental analysis, FT-IR, TGA and magnetic susceptibility measurements; the HMBC, DEPT, ¹H and ¹³C NMR spectra of the LH₂ ligand were also reported. The electrochemical property of the complexes was investigated in DMSO by cyclic voltammetry at 200 mV s⁻¹ scan rate. The cyclic voltammetric measurements clearly indicated that Co(LH)₂·2H₂O complex differs from the Ni(LH)₂ and Cu(LH)₂ complexes upon the exhibition of quasi-reversible one-electron transfer reduction process in the negative region instead of an irreversible process.     

The crucial importance of chemistry in the structure-function link: manipulating hydrogen bonding in iron-containing superoxide dismutase

Fe-containing superoxide dismutase's active site Fe is coordinated by a solvent molecule, whose protonation state is coupled to the Fe oxidation state. Thus, we have proposed that H-bonding between glutamine 69 and this solvent molecule can strongly influence the redox activity of the Fe in superoxide dismutase (SOD). We show here that mutation of this Gln to His subtly alters the active site structure but preserves 30% activity. In contrast, mutation to Glu otherwise preserves the active site structure but inactivates the enzyme. Thus, enzyme function correlates not with atom positions but with residue identity (chemistry), in this case. We observe strong destabilization of the Q69E-FeSOD oxidized state relative to the reduced state and intermediate destabilization of oxidized Q69H-FeSOD. Indeed, redox titrations indicate that mutation of Gln69 to His increases the reduction potential by 240 mV, whereas mutation to Glu appears to increase it by more than 660 mV. We find that this suffices to explain the mutants' loss of activity, although additional factors may also contribute. The strongly elevated reduction potential of Q69E-FeSOD may reflect reorganization of the active site H-bonding network, including possible reversal of the polarity of the key H-bond between residue 69 and coordinated solvent.     

Effects of endurance training and acute exhaustive exercise on antioxidant defense mechanisms in rat heart

We investigated whether 8-week treadmill training strengthens antioxidant enzymes and decreases lipid peroxidation in rat heart. The effects of acute exhaustive exercise were also investigated. Male rats (Rattus norvegicus, Sprague-Dawley strain) were divided into trained and untrained groups. Both groups were further divided equally into two groups where the rats were studied at rest and immediately after exhaustive exercise. Endurance training consisted of treadmill running 1.5 h day(-1), 5 days week(-1) for 8 weeks. For acute exhaustive exercise, graded treadmill running was conducted. Malondialdehyde level in heart tissue was not affected by acute exhaustive exercise in untrained and trained rats. The activities of glutathione peroxidase and glutathione reductase enzymes decreased by both acute exercise and training. Glutathione S-transferase and catalase activities were not affected. Total and non-enzymatic superoxide scavenger activities were not affected either. Superoxide dismutase activity decreased by acute exercise in untrained rats; however, this decrease was not observed in trained rats. Our results suggested that rat heart has sufficient antioxidant enzyme capacity to cope with exercise-induced oxidative stress, and adaptive changes in antioxidant enzymes due to endurance training are limited.     

Local delivery of chitosan/VEGF siRNA nanoplexes reduces angiogenesis and growth of breast cancer in vivo

Vascular endothelial growth factor (VEGF) is the important angiogenic factor associated with tumor growth and metastasis in a wide variety of solid tumors. The aim of this study is to investigate the tumor suppressive effect of chitosan/small interfering RNA (siRNA)-VEGF nanoplexes in the rat breast cancer model. Chitosan/siRNA nanoplexes (siVEGF-A, siVEGFR-1, siVEGFR-2) and NRP-1 were prepared in a 15 to1 ratio and injected (intratumorally) into the breast-tumor-bearing Sprague-Dawley rats. Tumor volumes were measured during 21 days. To investigate the effect of chitosan/siRNA nanoplexes on VEGF expression in tumors, VEGF was analyzed with immunohistochemistry and western blotting. The mRNA levels of VEGF in tumor samples were determined with real-time PCR (RT-PCR). After siRNA treatment, a marked reduction in tumor volumes was measured in complex-injected rats (97%). Free siRNA injection showed lower tumor inhibition. Reduction of VEGF protein was also shown with western blotting and immunohistochemistry. Similar results were obtained with RT-PCR also. These results indicate that the chitosan/siRNA targeting to VEGF nanoplexes have a remarkably suppressive effect on VEGF expression and tumor volume in breast cancer model of rats.     

Polyubiquitin Is Required for Growth, Development and Pathogenicity in the Rice Blast Fungus Magnaporthe oryzae

Protein ubiquitination, which is highly selective, regulates many important biological processes including cellular differentiation and pathogenesis in eukaryotic cells. Here, we integrated pharmacological, molecular and proteomic approaches to explore the role of ubiquitination in Magnaporthe oryzae, the leading fungal disease of rice world-wide. Inhibition of ubiquitin-mediated proteolysis using the 26S proteasome inhibitor, Bortezomib, significantly attenuated conidia germination, appressorium formation and pathogenicity in M. oryzae. Gene expression analysis revealed that many genes associated with protein ubiquitination were developmentally regulated during conidia germination. Only a few, including a polyubiquitin encoding gene, MGG_01282, were more abundantly expressed during appressorium formation and under nitrogen starvation. Targeted gene deletion of MGG_01282, in addition to a significant reduction in protein ubiquitination as determined by immuno blot assays, resulted in pleiotropic effects on M. oryzae including reduced growth and sporulation, abnormal conidia morphology, reduced germination and appressorium formation, and the inability to cause disease. Mutants were also defective in sexual development and were female sterile. Using mass spectrometry, we identified 63 candidate polyubiquitinated proteins under nitrogen starvation, which included overrepresentation of proteins involved in translation, transport and protein modification. Our study suggests that ubiquitination of target proteins plays an important role in nutrient assimilation, development and pathogenicity of M. oryzae.     

Effects of sugammadex on immunoreactivity of calcineurin in rat testes cells after neuromuscular block: a pilot study

Reversal of neuromuscular blockage induced by steroidal neuromuscular blocking agents such as rocuronium can be achieved using normal dose of sugammadex, which has been shown to be very effective for such reversal. In this study, we determined the effects of sugammadex on calcineurin immunoreactivity by examining the histopathological and histochemical structure of rat testis cells after neuromuscular blockage. Moreover, the regional distribution levels of calcineurin immunopositive testes cells were investigated. Eighteen adult male, Sprague–Dawley rats were divided into one control and two study groups. Study groups 1 and 2 rats received sugammadex at doses of 16 and 96 mg kg⁻¹ i.v., respectively, after rocuronium treatment (mg kg⁻¹ i.v.). The control group received intravenous 0.9% NaCl 1 ml. i.v without any drug. Our study demonstrates that sugammadex is safe and effective for reversal of rocuronium effects in rats, as well as in other animals and humans. Furthermore, histopathological examination indicates that high levels of sugammadex–rocuronium complexes accumulate a little in testis tissue. We found that rocuronium-sugammadex complexes were remained in circulation for a long time resulting in a decrease in interstitial space, testis size, germ cell numbers and Leydig cell numbers. Calcineurin immunoreactivity was higher in the experimental groups than the control group due to increase of calcium level. The results suggest that sugammadex–rocuronium complexes are cause histopathological and immunohistochemical changes in testis interstitial tissues, as well as changes in sperm density and germ cell number.     

Cadmium Reduces Contractile Responses of Rat Duodenum, In Vitro

The present study focuses on the effects of cadmium in drinking water on rat duodenum contractility, in vitro. Two experimental groups (n = 10 each) were administered with 15 ppm cadmium chloride in their drinking water for 1 and 2 months, respectively. Two separate groups (n = 10 each) were maintained in similar conditions but received no cadmium and acted as controls. After the experimental period, the duodenal segment responses were measured by means of an isotonic transducer. The average peak amplitude of acetylcholine-induced contractions was significantly decreased in both cadmium-treated groups. Incubation of duodena from the cadmium-free controls with atropine or in a calcium-free medium resulted in reduced contractility that was undistinguishable from that of the cadmium-treated rats. Cadmium reduces the contractile response apparently by directly or indirectly decreasing cellular calcium influx.