Monomeric structure of an active form of bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.

Discovery of non-competitive thrombin inhibitor derived from competitive tryptase inhibitor skeleton: Shift in molecular recognition resulted from skeletal conversion of carboxylate into phosphonate.

A novel series of terminal and internal phosphonate esters based on our previously developed aryl carboxylate-type tryptase selective inhibitor 1 was synthesized. The potency of these synthesized compounds was assessed in vitro with an enzyme inhibition assay using three available serine proteases, that is, tryptase, trypsin, and thrombin. The internal phosphonate derivative 6 showed potent thrombin inhibitory activity with an IC50 value of 1.0 µM, whereas it exhibited no or only weak tryptase and trypsin inhibition at 10 µM. The Lineweaver-Burk plot analysis indicates that the inhibition pattern of thrombin with 6 is non-competitive in spite of the fact that the lead carboxylate compound 1 is competitive inhibitor. Therefore, the skeletal conversion of the carboxylate into a phosphonate alters the mode of molecular recognition of these inhibitors by thrombin.

Hepatoprotective effect of Arctium lappa root extract on cadmium toxicity in adult Wistar rats.

This study was performed to determine the effects of Arctium lappa (Al) to protect against cadmium damage in the rat liver. Male rats received a single i.p. dose of CdCl2 (1.2 mg/kg body weight (BW)) with or without Al extract administered daily by gavage (300 mg/kg BW) for 7 or 56 days. After 7 days, Al caused plasma transaminase activity to diminish in groups Al (glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT)) and CdAl (GPT). After 56 days, GOT and GPT plasma activities were reduced in the Cd group. No alteration in plasma levels of creatinine, total bilirubin, and total protein were observed. GOT liver activity increased in the Cd group. No alteration was observed in superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), and malondialdehyde (MDA) dosage. In the Cd group, hepatocyte proportion decreased and sinusoid capillary proportion increased. In the Al and CdAl groups, the nuclear proportion increased and the cytoplasmic proportion decreased. The hepatocyte nucleus density reduced in Cd and increased in the Al group. After 56 days, there was no alteration in the Cd group. In Al and CdAl groups, the nuclear proportion increased without cytoplasmic proportion variation, but the sinusoid capillary proportion was reduced. The hepatocyte nucleus density decreased in the Cd group and increased in the Al and CdAl groups. In conclusion, the liver function indicators showed that A. lappa protected the liver against cadmium toxicity damage.

Naringin inhibits tumor growth and reduces interleukin-6 and tumor necrosis factor α levels in rats with Walker 256 carcinosarcoma.

The flavonoid naringin is a polyphenolic compound that naturally occurs in citrus. Patients with cancer generally present features of malnutrition and cachexia. Levels of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) are raised in patients with cancer. This study was designed to analyze the in vivo effect of naringin in the therapeutic treatment of rats bearing Walker 256 carcinosarcoma (W256). Rats were treated intraperitoneally with different doses of naringin (10, 25 and 35 mg/kg), for 50 days. At 25 mg/kg, naringin inhibited tumor growth by ~75%. With this treatment, TNF-α and IL-6 levels decreased (p<0.05) in comparison with the control. In addition, two rats presented complete tumor regression. Inhibition of tumor growth, survival increase and the reduction of TNF-α and IL-6 levels in rats bearing W256 treated with naringin strongly suggest that this compound has potential as an anticarcinogenic drug.

Discovery of diphenylmethane analogs as anti-bovine diarrhea viral agents.

Based on antiviral screening of our diphenylmethane derivatives prepared as steroid substitutes, we identified a 1,1-diphenylcyclobutane analog (9) and two diethyldiphenylsilane analogs (12 and 13) as superior lead compounds with potent anti-bovine viral diarrhea virus (BVDV) activity, having 50% effective concentration (EC(50): based on reduction of BVDV replication-induced cell destruction) and 50% cytotoxic concentration (CC(50): based on reduction of viable cell number) values of 6.2-8.4 microM and >100 microM, respectively, in Madin-Darby bovine kidney (MDBK) cells infected with BVDV.

Saline-irrigated radiofrequency ablation electrode with external cooling.

INTRODUCTION: Open flush, irrigated ablation electrodes may improve the safety of radiofrequency catheter ablation by preventing protein aggregation and coagulum formation. This is particularly important in left-sided procedures like catheter ablation of atrial fibrillation. Electrode cooling and the inherent loss of temperature feedback, however, grossly reduce the ability to monitor tissue heating. Intimate contact may not be recognized and the delivery of nominal RF power levels may then lead to excessive tissue heating, steam explosions, and even tamponade. METHODS AND RESULTS: Standard, open flush, irrigated catheters (Sprinklr, Medtronic Inc, Minneapolis, MN) were modified by thermally insulating the irrigation channels inside the ablation electrode. Using the thigh muscle preparation, multiple lesions were created with standard and modified catheters using 60 s, 20-50 Watt applications and a constant saline flush rate of 20 cc/min. A total of 57 lesions were created on five thigh muscles of three dogs. Lesion dimensions were not significantly different between both types of catheter, but the maximum electrode temperature rise during ablation was significantly higher with the modified catheter. Insulation of the irrigation channels improved the correlation coefficient between maximum electrode temperature rise and lesion volume from 0.38 (ns) to 0.62 (P < 0.001). CONCLUSION: Thermal insulation of the irrigation channels facilitates temperature feedback during radiofrequency ablation and controllability of lesion formation.

Influence of protein phosphatase inhibitors on HL60 cells death induction by dehydrocrotonin.

Oxidative stress can be involved in several cellular responses, such as differentiation, apoptosis and necrosis. Dehydrocrotonin (DCTN, diterpene lactone) from Croton cajucara, Brazilian medicinal plant, slightly induced NBT-reducing activity. In presence of protein phosphatase inhibitors significant differentiation of HL60 cells was observed. Flow cytometry analysis demonstrated that apoptosis was induced when the cells were treated with okadaic acid (OKA) and plus trans-dehydrocrotonin (t-DCTN) this effect was two-fold increased. Unlike, when the cells were treated only with t-DCTN, necrosis was observed. On the other hand, the necrosis induced by t-DCTN could be due to oxidative stress, revealed by increase of GSH content. Therefore, this differentiation pathway involves the modulation of protein phosphatases and this inhibition promotes the t-DCTN action on apoptosis induction.

Cytotoxic effect of the diterpene lactone dehydrocrotonin from Croton cajucara on human promyelocytic leukemia cells.

Diterpenes exhibit potent antineoplastic properties against human and murine carcinoma cell lines. trans-Dehydrocrotonin from Croton cajucara, a Brazilian medicinal plant, is a nor-diterpene with antiulcerogenic activity. In this work, we examined the effect of trans-dehydrocrotonin (t-DCTN) on the vitality of HL60 cells by assessing the MTT reduction, protein content and phosphatase activity of these cells. Protein quantification indicated that t-DCTN reduced the number of cells with an IC50 of 500 microM; mitochondrial function (MTT reduction), was also inhibited (IC50 = 300 microM), when the cells were treated for 24 h. In contrast, when the cells were treated with this lactone in the initial plating and cultured for 96 h, t-DCTN was more toxic for all parameters analyzed: MTT and phosphatase activity (IC50 = 180 microM) and protein content (IC50 = 150 microM). The flavonoid utilized as positive control myricetin and the following IC50 values were obtained after 24 h of treatment: 300 and 192 microM for protein content and MTT reduction, respectively. According to the chemical characteristics of both compounds, the cytotoxic effect of t-DCTN could be explained through two mechanisms: adduct formation with DNA and proteins and/or oxidative stress induction.