Expression profiling of microRNAs in RAW264.7 cells treated with a combination of tumor necrosis factor alpha and RANKL during osteoclast differentiation.

BACKGROUND AND OBJECTIVE: Tumor necrosis factor alpha (TNF-α), a cytokine involved in the pathogenesis of periodontal disease, induces osteoclast differentiation and indirectly promotes alveolar bone resorption. We investigated TNF-α-regulated osteoclast differentiation, focusing on microRNAs. MicroRNAs are small, noncoding RNAs that are involved in various biological processes, including cellular differentiation, proliferation and apoptosis. Aside from miR-21, miR-155 and miR-223, the identities of the microRNAs that play roles in osteoclast differentiation are unknown. Notably, no previous studies have reported the expression profiling of microRNAs during TNF-α-regulated osteoclast differentiation. MATERIAL AND METHODS: We used microarrays to screen the levels of expression of mature microRNAs in RAW264.7 cells treated with a combination of TNF-α and RANKL, or RANKL alone for 0, 24 or 82 h during osteoclast formation. We validated the results of the microarray analyses through quantitative RT-PCR analyses of representative microRNAs in RAW264.7 cells and murine bone marrow macrophages. RESULTS: During osteoclast formation, the expression of 44 mature microRNAs differed by more than twofold between untreated cells and cells treated with a combination of TNF-α and RANKL, and the expression of 52 mature microRNAs differed upon RANKL treatment. According to quantitative RT-PCR analyses, miR-378 was upregulated and miR-223 was downregulated during osteoclast formation. Furthermore, miR-21, miR-29b, miR-146a, miR-155 and miR-210 were highly expressed during osteoclast differentiation in TNF-α/RANKL-treated cells compared with RANKL-treated cells. CONCLUSIONS: These results suggest that miR-223 and miR-378 may play important roles in osteoclastogenesis, and that miR-21, miR-29b, miR-146a, miR-155 and miR-210 are involved in TNF-α-regulated osteoclast differentiation.

Impact of Smoking Cessation in Donor Candidates for Living Donor Liver Transplantation.

BACKGROUND: The relationship between smoking cessation and weight gain is well recognized. Examining the link between smoking cessation and weight gain in donor candidates for living donor liver transplantation (LDLT) is an important topic because of the influence of weight gain on the liver. This study assessed body weight (BW) changes after smoking cessation in donor candidates for LDLT. METHODS: The 27 donor candidates were retrospectively analyzed. The smoking status was determined based on questionnaires administered at the initial presentation, and the candidates were divided into 2 groups: recent quitters and nonsmokers. The changes in BW were compared between the groups. RESULTS: The recent quitters group included 10 (37.0%) candidates, and the nonsmokers group included 17 (63.0%). In the nonsmokers group, 1 candidate had gained weight since the initial presentation. In contrast, in the recent quitters group, 70.0% of candidates had gained weight since the initial presentation (P < .01). The change in BW from the initial presentation was greater in recent quitters than in nonsmokers (+1.6 kg [+2.4%] vs -0.5 kg [-0.9%]; P < .01). Two candidates in the recent quitters group gained ≥ 5 kg [8%] of weight. One of these 2 candidates was judged to be in a donor-inadequate status because of the appearance of fatty liver. CONCLUSIONS: Weight gain due to smoking cessation was observed in donor candidates for LDLT. The amount of weight gain after smoking cessation is highly individualized, so everyone concerned with LDLT must be alert to its potential development.

Alpha 1 adrenoceptor mediated signal transduction in neonatal rat ventricular myocytes: effects of prolonged hypoxia and reoxygenation.

STUDY OBJECTIVE: The aim was to study the effects of prolonged hypoxia and reoxygenation on alpha 1 adrenoceptors and inositol phosphate accumulation in neonatal rat ventricular myocytes maintained in culture for 6-8 d. DESIGN: Neonatal rat ventricular myocytes were subjected to 2 h hypoxia followed by 2 h reoxygenation. Cells were harvested at various times during hypoxia and after reoxygenation and measurements of alpha 1 adrenoceptor density and affinity and determinations of basal and (-)-noradrenaline stimulated inositol phosphate accumulation were carried out. EXPERIMENTAL MATERIAL: A neonatal rat ventricular myocyte preparation almost completely free of contaminating non-myocytes was used. Cells were grown in serum containing medium for 5 d before experiments were performed. alpha 1 Adrenoceptors were measured using the radioligand 125I-HEAT and inositol phosphates were measured by anion exchange chromatography after incubation with 1 microM (-)-noradrenaline for 5 min. MEASUREMENTS AND MAIN RESULTS: Hypoxia resulted in an increase in alpha 1 adrenoceptor density which was reversed by reoxygenation. There were no changes in antagonist affinity. (-)-Noradrenaline stimulated inositol phosphate production was increased at 1 h hypoxia but declined to control levels after 2 h hypoxia, while basal levels increased significantly at this time. This pattern was similar for all inositol phosphates measured: inositol-1-phosphate, inositol bisphosphate, and the putative second messenger, inositol trisphosphate. Displacement by (-)-noradrenaline of 125I-HEAT binding was significantly shifted to the right after 2 h hypoxia. CONCLUSIONS: Prolonged hypoxia in neonatal rat ventricular myocytes increases alpha 1 adrenoceptor density without change in antagonist affinity. Inositol phosphates follow a biphasic response, increasing after 1 h and decreasing after 2 h hypoxia in response to (-)-noradrenaline stimulation. This second messenger response and the rightward shift of the (-)-noradrenaline displacement curve suggests that after 2 h hypoxia there is a decrease in agonist affinity for the alpha 1 adrenoceptor consistent with uncoupling of the alpha 1 adrenoceptor from its effector.

Role of alpha 1-adrenoceptor activity in progression of cardiac hypertrophy in guinea pig hearts with pressure overload.

To elucidate the role of alpha 1- and beta-adrenergic activities in pressure overload hypertrophy, changes of alpha 1- and beta-adrenoceptors were measured by radioligand binding assay, and the preventive effects of alpha 1- and beta-adrenoceptor blockade on cardiac hypertrophy were assessed in guinea pigs after aortic banding. Five days after banding, dry weight of left ventricle had not increased, though wet weight increased due to marked intercellular oedema. In this period, the maximum binding capacity of [3H] prazosin increased to 31.1 (SEM 2.2) fmol.mg-1 from (sham operation) 17.0(2.1) fmol.mg protein-1, p less than 0.01, whereas the maximum binding capacity of [3H]dihydroalprenolol did not increase: 143(16) fmol.mg-1 (banded) v 153(13) fmol.mg-1 (sham). Three weeks after aortic banding, the maximum binding capacity of both ligands increased to 45.6(5.5) fmol.mg-1 and 232(21) fmol.mg-1, respectively, accompanied by a significant increase in left ventricular dry weight, from 0.46(0.02) mg.g-1 (sham) to 0.62(0.08) mg.g-1 (banded), p less than 0.01. Continuous subcutaneous administration of the alpha 1-blocker bunazosin (0.1 mg.kg-1.d-1) significantly attenuated the increase in left ventricular dry weight whereas the beta-blocker propranolol (5 mg.kg-1.d-1) did not: 0.55(0.03) v 0.66(0.04) mg.g-1 respectively, after 3 weeks. These results show that pressure overload elicited an increase in myocardial alpha 1-adrenoceptors before the onset of cardiac hypertrophy, and that an alpha 1-blocker could prevent the development of hypertrophy in the pressure overloaded heart.

An increase in myocardial beta-adrenoceptors to compensate for postischaemic dysfunction following coronary micro-embolisation in dogs.

This study examined whether beta-adrenoceptors increase in number during recovery from prolonged myocardial stunning and whether they compensate for lack of physiological response to beta-adrenergic stimulation in this abnormality. The left coronary artery was embolished in anaesthetised dogs with non-labelled microspheres (15 +/- 1 micron; 1.2 X 10(6).kg-1 body weight). Haemodynamic studies were performed before (control) and 24 h and 1 week after embolisation, in the conscious state. Myocardial noradrenaline content, plasma catecholamine concentrations and the density of beta-adrenoceptors (Bmax) were also assessed at three study intervals. At 24 h after embolisation, both systolic and diastolic cardiac function was significantly depressed. The inotropic response to isoprenaline was preserved, but the response to forskolin was markedly depressed. One week after embolisation, resting systolic function was restored to control levels and histological examination showed absence of myocardial necrosis. Although plasma noradrenaline concentration had returned to normal, myocardial noradrenaline content had decreased by 36% and the density of beta-adrenoceptors had increased by 48%. Myocardial relaxation was still impaired and the inotropic response to forskolin was also still depressed, whereas the response to isoprenaline was normal. Moreover, the down regulation of the increased beta-adrenoceptors by isoprenaline infusion for 24 h unmasked the latent systolic dysfunction. These results indicate that the density of beta-adrenoceptors increases during the recovery process from prolonged myocardial stunning and that this increase may compensate, at least in part, for impairment of the inotropic mechanism distal to the beta-adrenoceptors.

Radiosensitization in vitro and in vivo by 3-nitrotriazoles.

A series of 3-nitro-1,2,4-triazole derivatives bearing various types of side chain (R) at the N1-position (AK-2000 series) were synthesized and their radiosensitizing effect and toxicity in vitro and in vivo were investigated, in comparison with those of Misonidazole (MISO), SR-2508, and RSU-1069. Of the fifteen 3-nitrotriazoles tested, all had sensitizing effects in vitro on hypoxic V79 cells. Also, all but one had definite effects on solid EMT6/KU and SCCVII tumors in vivo. For many of the triazole compounds, the degree of radiosensitization in vitro and in vivo appeared identical. However, they were generally less efficient, both in vitro and in vivo, than the corresponding 2-nitroimidazoles, whereas their aerobic cytotoxicity and toxicity to mice (LD50/7) were comparable to those of the 2-nitroimidazoles. Considering the sensitizing effect and toxicity, AK-2123 (R = CH2CONHC2H4OCH3) may be as useful as MISO, but none of the triazoles have been proved to be superior to SR-2508.

Importance of tumor affinity of nitroazoles in hypoxic radiosensitization.

In vitro and in vivo sensitizing activities of a variety of nitroazole derivatives including misonidazole (MISO), SR-2508, and RSU-1069 were correlated by the aid of pharmacokinetic measurements of the drug uptake in animal solid tumors. The sensitizer enhancement ratio in vivo (SERvivo) on solid tumors increased linearly with the square root of administrated dose (Ds). The specific activity (A) in vivo of nitroazoles was evaluated from the square-root empirical relationship, SERvivo = 1.00 + A D1/2S. The intratumor concentration of nitroazoles at a given time t after administration was in proportion to the DS, in which the proportional constant was defined as the tumor-affinity factor FT,t. The absolute molar activity alpha M defined by A(M/FT,t)1/2, where M is the molecular weight of nitroazoles, showed a linear relationship with the SER in vitro (SERvitro) at 1 mM of sensitizers. The sensitizer dose required to achieve an SERvivo of 1.5 (DS,1.5) decreased and thus the overall sensitizing efficiency on animal solid tumors increased as the FT,t became greater.

Characteristics of fluorinated nitroazoles as hypoxic cell radiosensitizers.

Types of 2-nitroimidazoles and 3-nitro-1,2,4-triazoles bearing one or two fluorine atoms on their side chains were synthesized to evaluate their physicochemical properties, radiosensitizing effects, and toxicity. The reduction potential of the compounds containing one fluorine was similar to that of misonidazole (MISO), whereas that of the difluorinated compounds was slightly higher. Both mono- and difluorinated compounds had an in vitro sensitizing activity comparable to or slightly higher than that of MISO. The fluorinated 3-nitrotriazoles were almost as efficient as the 2-nitroimidazoles with the same substituent. In vivo, some of the compounds were up to twice more efficient than MISO, whereas others were as efficient as MISO. Toxicity in terms of LD50/7 in mice was quite variable depending on the side-chain structure; the amide derivatives were less toxic than MISO, whereas the alcohol and ether derivatives were more toxic. In view of the radiosensitizing effect and toxicity in vivo, at least one compound, KU-2285 (a 2-nitroimidazole with an N1-substituent of: CH2CF2CONHCH2CH2OH) has been found to be as useful a hypoxic cell sensitizer as SR-2508.

A fluorinated 2-nitroimidazole, KU-2285, as a new hypoxic cell radiosensitizer.

To develop new hypoxic cell radiosensitizers, we incorporated fluorine atoms into the side chain of the 2-nitroimidazole. Of the resulting compounds, KU-2285 (a 2-nitroimidazole with an N1-substituent of CH2CF2CONHCH2-CH2OH) was considered the most useful as a hypoxic cell radiosensitizer. In this study, its in vivo radiosensitizing activity and acute toxicity were compared with those of etanidazole. The reduction potentials of KU-2285 and etanidazole were -0.96 V and -1.05 V vs Ag/Ag+ in N,N-dimethylformamide, respectively, and their respective octanol/water partition coefficients were 0.25 and 0.040. The in vivo radiosensitizing activity of KU-2285 was found to be similar to that of etanidazole at the same administration dose when assayed by an in vivo-in vitro assay, a growth delay assay, and a tumor control assay using SCC VII tumor or transplanted mammary tumor in C3H/He mice. Although the radiosensitizing activity of etanidazole was reduced when it was administered orally, there was no significant difference in the radiosensitizing activity of KU-2285 whether it was administered intravenously, intraperitoneally, or orally. The acute toxicity measured as the LD50/7 in 8-week-old female C3H/HeJ mice was found to be 2.4 g/kg (intravenously), 2.1 g/kg (intraperitonealy), and 4.25 g/kg (orally) for KU-2285, whereas it was 4.75 g/kg (intravenously) for etanidazole.

Effects of reserpine and adenosine agonist on ?(2)-adrenergic receptor of rat vas deferens examined with [(3)H]yohimbine and [(3)H]clonidine.

The changes of [(3)H]yohimbine and [(3)H]clonidine binding sites in rat vas deferens on treatments with adenosine receptor agonists (2-chloroadenosine, adenosine) or reserpine were examined. Treatment with adenosine agonist in vitro increased [(3)H]clonidine binding sites but had no influence on affinity and number of binding sites of ?(2)-antagonist, [(3)H]yohimbine. Amount of [(3)H]yohimbine binding sites was found to be higher than that of [(3)H]clonidine with or without the treatment. Inhibition curves of ?(2)-agonists, clonidine and norepinephrine, on [(3)H]yohimbine binding were less than unity though ?(2)-antagonist inhibited with about 1.0 of n(H). The treatment with adenosine agonist reduced the IC(50) value of agonists on the [(3)H]yohimbine binding but had no influence on the inhibitory effect of antagonist. These effect of adenosine agonists was completely blocked by theophylline. Accordingly it was considered that activation of adenosine receptor caused configurational change in ?(2)-adrenergic receptor from low affinity state for agonist to the high affinity state, though both states had same affinity for antagonist. On the other hand, treatment with reserpine in vivo increased the affinity of clonidine for ?(2)-adrenergic receptors and also increased the amount of the ?(2)-receptors.

Cardiac M2 receptors consist of two different types, both regulated by GTP.

Cardiac muscarinic receptors are predominantly M2 receptors, and have three agonist binding sites (super-high(SH), high(H) and low(L) affinity agonist binding sites). Treatment of cardiac membranes with 50 nM propylbenzilyl choline mustard (PrBCM) caused 88% loss of binding sites for [3H]QNB. Carbamyl choline (CCh) inhibits this alkylation dose dependently and, theoretically, generates uneven alkylation of multiple agonist binding sites. Pretreatment of the membranes with 50 nM PrBCM and 0.5 mM CCh resulted in almost complete disappearance of L sites with similar degrees of conservation of H sites and SH sites. In these pretreated membranes, guanine nucleotide and sulfhydryl reagent caused a change in the ratio of residual SH and H sites but not of L sites though previous studies showed that, in intact membranes, these reagents affected the ratio of SH and L sites without significantly changing that of the H site. These results indicate the existence of two equilibria regulated by guanine nucleotide and sulfhydryl reagent in cardiac muscarinic receptors: one between SH and H sites and the other between H and L sites. The participation of GTP binding protein(s) in all cardiac muscarinic responses is suggested.

Multiple agonist binding sites of muscarinic acetylcholine receptors and their relation to the negative inotropic action of agonists in guinea-pig heart.

The Kd values of the multiple agonist binding sites in cardiac muscarinic receptors (mAChR) and pD2 values for negative inotropic actions were determined independently and their relation was examined. The guinea-pig cardiac mAChR is known to have three agonist binding sites (super-high (SH), high (H) and low (L) affinity agonist binding sites) for carbachol (CCh). Pilocarpine (Pilo) and oxotremorine (Oxo) distinguished two sites (higher (Ho/p) with pKd of 5.88 and 8.20, respectively, and lower (Lo/p) affinity agonist binding sites with pKd of 5.08 and 6.17, respectively). The effects of guanine nucleotide and sulfhydryl reagent indicated that the Ho/p site corresponded with the SH site for carbachol, and the Lo/p site with the H + L sites for carbachol. The pD2 values of CCh, Pilo and Oxo for negative inotropic actions on autocontraction of right atria were 5.38, 5.30 and 6.80, respectively. The pD2 values of CCh and Oxo on electrically stimulated contraction of left atria in the presence of isoproterenol were 5.80 and 6.46, respectively, thus being closely related to H or Lo/p agonist binding sites of mAChR.

Effects of phenolic compounds on reactions involving various organic radicals.

Investigation of effects produced by 26 various phenol and diphenol derivatives, including industrial and natural antioxidants (ionol, bis-phenol 2246, alpha-tocopherol), on final product yields of radiation-induced free-radical processes involving peroxyl, alkyl, alpha-hydroxyalkyl and alpha,beta-dihydroxyalkyl radicals has been performed. Ionol and bis-phenol 2246 have been shown to be more effective than alpha-tocopherol or diphenol derivatives in suppressing hydrocarbon oxidation processes. At the same time, alpha-tocopherol and its water-soluble analogues, as well as diphenol-based substances, are more effective than phenol derivatives in regulating various homolytic processes involving carbon-centered radicals. This fact can be accounted for by taking into consideration the contribution to formation of the final product set and the respective yields made by semiquinone radicals and compounds with quinoid structure arising in the course of homolytic transformations in systems containing diphenol derivatives.

Effect of a hypoxic radiosensitizer, AK 2123 (Sanazole), on yeast Saccharomyces cerevisiae.

Sanazole/DNA repair/Hypoxic radiosensitization/DNA polymerases/Saccharomyces cerevisiae Yeast Saccharomyces cerevisiae can exist in two physiological states, namely anaerobic and aerobic. They differ in their response to gamma- radiation and radiomodification. We report hereon our results concerning radiosensitization by Sanazole (AK-2123), a well-known hypoxic radio sensitizer, whose mechanism of action has been studied extensively. The results have revealed that Sanazole (1 mM) when present during irradiation could specifically sensitize wild-type anaerobic yeast cells with a DMF of 2.4. In a radiation-sensitive mutant which lacks a DNA repair pathway specific for the recovery from gamma-radiation induced DNA damage, the extent of sensitization was considerably lower and the DMF was only 1.3. Studies on the liquid holding recovery of cells of both wild- type and rad52 yeast cells exposed to radiation in presence of Sanazole revealed that sensitization by Sanazole is due to a preferential increase in the DNA damage, and not by impairing DNA repair. This system thus holds promise for screening potential hypoxic chemical radiosensitizers.

Redox reactions of sanazole (AK-2123) in aqueous solutions: a pulse radiolysis study.

The redox chemistry of sanazole, an efficient hypoxic cell radiosensitizer, generally referred to as AK-2123, was studied by pulse radiolysis with eaq-, CO2-., 2-propanol radicals and CH2OH radicals. AK-2123 reacts with eaq-, CO2-. and 2-propanol radicals at almost diffusion-controlled rates, producing a nitro radical anion (lambda max = 290 nm) within a few microseconds. The decay kinetics of the radical anion was independent of the pH. The radical anion reacts with oxygen with a rate constant of 3.4 x 10(6) dm3 mol-1 s-1. An electron-transfer reaction was observed from the thymine radical anion to AK-2123. From redox equilibria with methyl viologen, the one-electron reduction potential of AK-2123 in aqueous solution, determined by pulse radiolysis, was estimated to be -0.33 +/- 0.02 V vs. NHE. Depletion of intracellular nonprotein thiols did not mitigate the radiosensitizing affect of the hypoxic radiosensitizer, AK-2123.

Radiosensitizer AK-2123 as modulating agent in the chemotherapy of experimental metastases.

Therapeutic effect of Cyclophosphamide (CPA) and radiosensitizer AK-2123 (AK) combination versus CPA alone in the same doses was investigated on transplanted LL carcinoma and B 16 melanoma. Antimetastatic efficacy of different doses of CPA and combined therapy was evaluated. Our data demonstrate that the effect of combined treatment by CPA at low uneffective doses (60 mg/kg, 40 mg/kg, 20 mg/kg at the 3rd and the 7th day after transplantation) and AK at low daily doses (1 mg/kg and 0.1 mg/kg for 3-9 days after transplantation) is equal or superior to the effect of CPA alone at the therapeutic dose (120 mg/kg).

Inhibitory effect of radiosensitizer AK-2123 on experimental hepatic metastases and Ca2+ active transport.

A triazole group radiosensitizer AK-2123 is shown to inhibit considerably the growth of hepatic metastases induced by the intrasplenic injection of colon adenocarcinoma cells in syngenic mice. Even an extremely low dose of the drug exhibits the antimetastatic effect. It is shown that AK-2123 injected at therapeutic dose inhibits active transport of calcium ions by the (Ca2+-Mg2+)-dependent ATP-ase. The antimetastatic effect of AK-2123 is suggested to be related, at least partially, to the inhibition of the active calcium transport.

Nitrotriazole AK-2123 enhances mitomycin C activity in mice bearing multidrug-resistant tumors.

The often observed cross resistance of multidrug-resistant (MDR) tumors to mitomycin C (MMC) is surprising, as these tumors are, as a rule, sensitive to alkylating drugs, and the mechanism of MMC activity is connected to alkylation of DNA. This study shows that nitrotriazole AK-2123 significantly enhances the sensitivity of MDR-strains of P388 mouse leukemia (developed and characterized by authors previously) to mitomycin C. The modulating effect is dependent on the initial sensitivity of resistant tumors to MMC which is correlated with the existence or absence of sorcin (cytosole Ca2+-binding protein) gene coamplification in MDR-amplicon. In agreement with authors' previous data about AK-2123 influence on active Ca2+-transport, it is supposed that the modulatory effect of radiosensitizer is at least partially dependent on this capacity. AK-2123 has no own antitumor effect on investigated tumors and cannot modify the sensitivity of the parent tumor P388 to MMC.

Modification of radio-thermo-chemotherapy by AK-2123 and hydralazine in tumor bearing mice.

The effects of chemical modifiers of hypoxic radiosensitizer, a 3-nitrotriazole derivative AK-2123 (200 mg/kg) before treatment, and vasodilator of hydralazine (HDZ; 5.0 mg/kg) after treatment on tumor growth of SCCVII of mice were investigated in the radio-thermotherapy combined with mitomycin C (MMC; 2.0 mg/kg) or adriamycin (ADM; 3 mg/kg). The tumor treated by 10 Gy alone (tumor doubling time = 7.5 days), MMC alone (6.9 days), and hyperthermia (43 degrees C, 30 min; HT) alone (8.0 days) showed a slight growth delay (control: 5.6 days). Prolonged growth delay (23.2 days) was observed by MMC-radio-thermotherapy (MMC-10Gy/HT) than that (12.4 days) by 10 Gy/HT. The modification of MMC-radio-thermotherapy by HDZ administered between 10 Gy and HT (MMC-10 Gy/HDZ/HT) resulted in the significant prolongation of tumor growth delay (31.7 days). AK-2123 administration before this treatment, (MMC-AK-2123)-10 Gy/HDZ/HT), enhanced a further tumor growth delay (37.6 days) which is equal to that by 50 Gy alone and resulted in the highest dose modifying factor (DMF) of 5.2. While modification of ADM-radio-thermotherapy by AK-2123 and HDZ, (ADM-AK-2123)-10 Gy/HDZ/HT, gave the equal tumor growth delay to that by 30 Gy alone (DMF = 3.1). These high efficacies of radio-thermo-chemotherapy modified by AK-2123 and HDZ may be caused by tumor blood flow reduction.