Living organisms have developed a wide variety of energy metabolism to survive within the specialized environments. There is a remarkable diversity in mitochondrial electron transport system, which might be potential targets for chemotherapy. Atovaquone, clinically used to treat malaria and pneumocystis pneumonia, is a specific inhibitor of Qo site in the cytochrome bc1 complex of Plasmodium falciparum and Pneumocystis jirovecii. Phytopathogenic fungus, Ascochyta viciae produces two antibiotics, ascochlorin and ascofuranone. Ascochlorin specifically binds to inhibit the electron transport of both Qi and Qo sites in cytochrome bc1 complex. Besides the unique respiratory inhibition, further investigation is in progress to elucidate the effects on cancer cells. On the other hand, ascofuranone specifically inhibits cyanide-insensitive trypanosome alternative oxidase, which is a sole terminal oxidase in the mitochondrion of Trypanosoma brucei, causative of African trypanosomiasis. In vivo study suggests that ascofuranone is a promising candidate for chemotherapeutic agents to treat African trypanosomiasis.
Mitochondrial permeability transition (PT) is the phenomenon in which the mitochondrial inner membrane becomes permeable to various solutes and ions. When PT is induced by Ca2+, cytochrome c is released from mitochondria into the cytosol where it then triggers subsequent steps of programmed cell death, apoptosis. Thus, the proteins that regulate PT and cytochrome c release could become druggable targets for various diseases. However, the mechanisms of PT and the release of cytochrome c have not yet been revealed. We previously showed that valinomycin, a potassium selective ionophore, also caused release of cytochrome c from mitochondria without inducing PT. This result indicates that cytochrome c could be released from mitochondria with or without induction of PT. In this study, to understand the difference of effects of valinomycin and Ca2+ on mitochondria, we examined what protein species are released from valinomycin- and Ca2+-treated mitochondria by LC-MS/MS. As a result, only the proteins located in the intermembrane space were found to be released from valinomycin-treated mitochondria, while those in both the intermembrane space and in the matrix were released from Ca2+-treated mitochondria. Furthermore, the protein releases by each reagent occurred not selectively but in a concentration-dependent manner. Based on these results, the permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes are discussed.
Parkinson's disease is a degenerative disorder of the central nervous system caused by selective dopamine-generating cell death, and oxidative stress and mitochondrial dysfunction are thought to be responsible for the onset of Parkinson's disease. While most cases of Parkinson's disease are idiopathic, 5-10% of cases are attributed to genetic factors. DJ-1 was first identified as an activated ras-dependent oncogene and later found to be a causative gene for a familial form of Parkinson's disease, PARK7. We and others found that DJ-1 plays roles in transcriptional regulation and anti-oxidative stress function, and loss of its function is thought to affect the onset of Parkinson's disease. DJ-1 is mainly located in the cytoplasma and nucleus and partially in mitochondria. When mice or mouse cells were treated with bisphenol A, an endocrine disruptor and inducer of reactive oxygen species, DJ-1 was translocated into mitochondria to maintain mitochondrial complex I activity. We also found that DJ-1 directly bound to and was co-localized with NDUFA4 and ND1, nuclear and mitochondrial DNA-encoding subunits of mitochondrial complex I, respectively, and that these associations were enhanced by oxidative stress. Furthermore, complex I activity was reduced in two types of DJ-1-knockdown NIH3T3 and HEK293 cells. These findings suggest that DJ-1 is an integral mitochondrial protein and maintains mitochondrial complex I activity to regulate mitochondrial homeostasis.
Mitochondrial dysfunction has been implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require the targeted delivery of therapeutic agents to mitochondria. This will likely be achieved through innovations in the areas of the nanotechnology of intracellular trafficking. Mitochondrial delivery systems for a variety of cargoes have been repored to date. However, only a limited number of approaches are available for delivering macromolecules directly to mitochondria. We previously reported on the construction of a MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. Using the green fluorescence protein as a model macromolecule in conjunction with analysis by confocal laser scanning microscopy, we were able to confirm the mitochondrial delivery of a macromolecule by the MITO-Porter. Moreover, we reported that the Dual Function MITO-Porter (DF-MITO-Porter) could efficiently deliver cargo to mitochondria, through endosomal and mitochondrial membranes via step-wise membrane fusion. Here, We will present our findings on the development of our mitochondrial drug delivery system, and discuss our attempts regarding mitochondrial gene delivery and therapy. Finally, We will discuss the potential use of mitochondrial drug delivery systems in mitochondrial medicine.
This review summarizes the results that were obtained by the author at School of Pharmaceutical Sciences and Graduate School of Biomedical Sciences, Nagasaki University with many co-authors as a memoir upon the author's retirement. The author has studied the development and application of sensitive determination methods for bio-related compounds mainly using fluorescence and/or chemiluminescence as the detection tool in instrumental analysis. As the most interesting bio-related compounds, the author mainly selected drugs of abuse and determined their presence in biological samples such as hair and urine. Also, the author focused on the development of new analytical reagents such as fluorescent and chemiluminescent reagents. Some of these were very useful and became commercially available. I hope that the developed methods would be useful and helpful in the pharmaceutical and forensic science fields.
Organic nitroxyl radical catalysts have recently attracted great attention because they realize efficient alcohol oxidation under mild and environmentally benign conditions. A representative of this class is 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO). We have also developed 2-azaadamantane N-oxyls (AZADOs) as highly efficient oxidation catalysts. These nitroxyl radicals are generally oxidized by a cooxidant to generate oxoammonium salts, which are active species for alcohol oxidation. In the oxidation systems presented in this paper, we focus on the differences between these two species in terms of oxidation state and counter anion. Herein, the effects of a counter anion of an oxoammonium species on its reaction selectivity are shown. On the basis of the control of the counter anion, we have developed catalytic oxidative rearrangement of tertiary allylic alcohols to β-substituted α,β-unsaturated carbonyl compounds. Moreover, we have developed novel useful oxidation systems utilizing a catalytic oxoammonium salt; namely, a one-pot oxidation of primary alcohols to carboxylic acids and an aerobic alcohol oxidation.
L-Glutamate (Glu) and γ-aminobutyric acid (GABA) has been thought to be an excitatory/inhibitory amino acid neurotransmitter in the mammalian central nervous system (CNS). Limited information is available in the literature with regard to an extracellular transmitter role of Glu and GABA in peripheral neuronal and non-neuronal tissues, whereas recent molecular biological analyses including ours give rise to a novel function for Glu and GABA as an autocrine and/or paracrine factor in a variety of cells derived from mesenchymal stem cells, in addition to other peripheral tissues including pancreas, adrenal, and pituitary glands. Emerging evidence suggests that Glu and GABA could play a dual role in mechanisms underlying maintenance of cellular homeostasis as a neurotransmitter in the CNS and as an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, therefore, we summarized the possible signaling by Glu and GABA as an extracellular signal mediator in mechanisms underlying maintenance of cellular homeostasis in mesenchymal stem cells, osteoblasts and chondrocytes.
Rhemium-186-1-hydroxyethylidene-1,1-diphosphonate (186Re-HEDP) has been used for the palliation of metastatic bone pain. However, delayed blood clearance and high gastric uptake of radioactivity have been observed upon injection, due to the instability of 186Re-HEDP. We designed, synthesized and evaluated a stable 186Re-mercaptoacetylglycylglycylglycine (MAG3) complex-conjugated bisphosphonate, [[[[(4-hydroxy-4,4-diphosphonobutyl)carbamoylmethyl] carbamoylmethyl]carbamoylmethyl]carbamoylmethanethiolate] oxorhenium(V) (186Re-MAG3-HBP). The stability of 186Re-MAG3-HBP and 186Re-HEDP in phosphate buffer were compared. No measurable decomposition of 186Re-MAG3-HBP occurred, while only approximately 30% of 186Re-HEDP remained intact 24 hours post-incubation. In biodistribution experiments, the radioactivity level of 186Re-MAG3-HBP in bone was significantly higher than that of 186Re-HEDP. Blood clearance of 186Re-MAG3-HBP was faster than that of 186Re-HEDP. In addition, the gastric accumulation of 186Re-MAG3-HBP radioactivity was lower. To evaluate the therapeutic effects of 186Re-MAG3-HBP, an animal model of bone metastasis was prepared. In the rats treated with 186Re-HEDP, tumor growth was comparable to that in untreated rats. In contrast, when 186Re-MAG3-HBP was administered, tumor growth was significantly inhibited. Bone pain was attenuated by treatment with 186Re-MAG3-HBP or 186Re-HEDP, but 186Re-MAG3-HBP tended to be more effective. These results indicate that 186Re-MAG3-HBP could be useful as a therapeutic agent of metastatic bone pain. Moreover, based on the similar concept, we designed, synthesized, and evaluated a 99mTc-6-hydrazinopyridine-3-carboxylic acid-conjugated bisphosphonate (99mTc-HYNIC-HBP) as a bone scintigraphic agent. 99mTc-HYNIC-HBP gave higher levels of radioactivity in bone than 99mTc-HMDP. There was no significant difference in clearance from blood between 99mTc-HYNIC-HBP and 99mTc-HMDP. Consequently, 99mTc-HYNIC-HBP showed a higher bone-to-blood ratio than 99mTc-HMDP. The findings indicate that 99mTc-HYNIC-HBP holds great potential for bone scintigraphy.
Pharmacokinetic studies of nanoparticles have provided significant information by which advanced delivery systems have been designed. On the other hand, pharmacokinetics of RNA interference (RNAi) effectors such as small interfering RNA (siRNA) is poorly understood. We have synthesized [18F]-labeled siRNA for a pharmacokinetic study using positron emission tomography (PET). PET is a noninvasive bioimaging modality applicable to a microdosing study that is designed to evaluate pharmacokinetics of drug candidates in human at an early stage of drug development. We have demonstrated that the resulting PET images clearly show the biodistribution of siRNA in mice. Naked [18F]-siRNA accumulates in the bladder immediately after intravenous administration due to rapid degradation. [18F]-siRNA formulated in cationic liposomes rapidly accumulates in the lungs due to positive surface charge of the cationic liposomes. [18F]-siRNA formulated in PEGylated liposomes shows long circulation in the bloodstream. Thus, the biodistribution of [18F]-siRNA is reflected by the characteristics of each liposomal biodistribution. Importantly, it has been demonstrated that accumulation of siRNA in tumors is able to be evaluated using [18F]-siRNA/PET. In addition, [18F]-siRNA/PET is also found to be useful to evaluate the effect of chemical modification of siRNA on its biodistribution. Cholesterol-conjugated [18F]-siRNA shows long half-life in the bloodstream and accumulates in the liver compared with naked [18F]-siRNA. Our findings indicate that the pharmacokinetic PET study of siRNA provides important insights into the development of RNAi medicine.
The products of proto-oncogene play critical roles in the development or maintenance of multicellular societies in animals via strict regulatory systems. When these regulatory systems are disrupted, proto-oncogenes can become oncogenes, and thereby induce cell transformation and carcinogenesis. To understand the molecular basis for development of the regulatory system of proto-oncogenes during evolution, we screened for ancestral proto-oncogenes from the unicellular choanoflagellate Monosiga ovata (M. ovata) by monitoring their transforming ability in mammalian cells; consequently, we isolated a Pak gene ortholog, which encodes a serine/threonine kinase as a ‘primitive oncogene’. We also cloned Pak orthologs from fungi and the multicellular sponge Ephydatia fluviatilis, and compared their regulatory features with that of M. ovata Pak (MoPak). MoPak is constitutively active and induces cell transformation in mammalian cells. In contrast, Pak orthologs from multicellular animals are strictly regulated. Analyses of Pak mutants revealed that structural alterations in the auto-inhibitory domain (AID) are responsible for the enhanced kinase activity and the oncogenic activity of MoPak. Furthermore, we show that Rho family GTPases-mediated regulatory system of Pak kinase is conserved throughout the evolution from unicellular to multicellular animals, but the MoPak is more sensitive to the Rho family GTPases-mediated activation than multicellular Pak. These results show that maturation of AID function was required for the development of the strict regulatory system of the Pak proto-oncogene, and support the potential link between the development of the regulatory system of proto-oncogenes and the evolution of multicellularity. Further analysis of oncogenic functions of proto-oncogene orthologs in the unicellular genes would provide some insights into the mechanisms of the destruction of multicellular society in cancer.
An initial loading procedure has been recommended to enable teicoplanin to promptly reach an effective serum concentration for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). The mean trough concentrations were 13.2 mg/L for patients with eradication of MRSA. I showed that the administration of ≥36 mg/kg during the first 3 days was effective in promptly obtaining a trough concentration target of ≥13 mg/L for the initial treatment of MRSA infections. The major adverse effect associated with linezolid treatment is reversible myelosuppression, primarily thrombocytopenia. I demonstrated that the incidence of linezolid-induced thrombocytopenia was higher among patients with renal failure than in patients with normal renal function. A statistically significant (p<0.01) and strong correlation (r=0.933) was observed between linezolid and creatinine clearance. A negative correlation (r=−0.567) was also shown between linezolid clearance and blood urea nitrogen. Voriconazole trough plasma concentration has been reported to correlate with hepatotoxicity. Logistic regression analysis revealed that the therapeutic range for the voriconazole trough concentration should be 2-4 mg/L. Nonlinear pharmacokinetic analysis suggested that voriconazole therapy should be initiated with a dose of 7.2-8.9 mg/kg/day for CYP2C19 wild type and 4.4-6.5 mg/kg/day for the non-wild type in patients. Anaemia and thrombocytopenia are major side effects of liposomal amphotericin B. I demonstrated that the doses of liposomal amphotericin B estimated to cause side effects of a low red blood cell count, anaemia and thrombocytopenia with 50% probability are 4.0, 3.3 and 3.0 mg/kg/day, respectively.
We adopted peer evaluation (mutual evaluation between students) for small group discussion (SGD) among first graders. The peer evaluation criteria were 5 grade scales for 5 fields: “preparation,” “remark,” “listening,” “activeness,” and “role.” A comparison with tutor evaluation clarified the validity of peer evaluation for summative evaluation. Although the average of peer evaluation (4.2 (4.0-4.4)) was higher than that of tutor evaluation (3.8 (3.7-4.1)) (p=0.0601, Mann-Whitney U test), the value of the correlation coefficient between peer evaluation and summative evaluation of SGD (average 0.35 (0.12-0.54)) was almost the same as that of the coefficient between tutor evaluation and summative evaluation of SGD (average 0.36 (0.24-0.42)) (p=0.6761, Mann-Whitney U test). Principal component analysis showed that the tutor could not evaluate “remark” and “listening” independently, while students evaluate “listening” independently from other evaluation criteria. The combination of peer and tutor evaluation may be multilateral evaluation for SGD. The questionnaire about peer evaluation for students showed that they recognized the value of peer evaluation and favorably accepted its use.
Currently, there is a need to reduce the occupational exposure of health care workers to anticancer drugs. Environmental contamination by anticancer drugs and subsequent exposure of health care workers are associated with vaporization of anticancer drugs. Furthermore, carcinomatous unpleasant odor is an additional problem to vaporized anticancer drugs in the field of clinical cancer therapy. We attempted to degrade vaporized anticancer drug and unpleasant odor using a photocatalyst. Cyclophosphamide or unpleasant odors (ammonia, formaldehyde, isovaleric acid, and butyric acid) were vaporized by heating in a closed chamber. Plates of photocatalyst coated with titanium dioxide were placed into the chamber and irradiated by light source. Vaporized cyclophosphamide in the chamber was recovered by bubbling the internal air through acetone and derivatized by trifluoroacetic anhydride for analysis by gas chromatographic-mass spectrometric assay. Vaporized odors were determined using a gas-detector tube, which changed color depending on the concentration. Following activation of the photocatalyst by a light source, the residual amounts of anticancer drug and unpleasant odor components were significantly decreased compared with when the photocatalyst was not activated without a light source. These results indicate that the photocatalysts can accelerate the degradation of vaporized anticancer drugs and odor components. Air-cleaning equipment using a photocatalyst is expected to be useful in improving the QOL of cancer patients experiencing carcinomatous unpleasant odor, and in reducing occupational exposure of health care workers to anticancer drugs.
The use of triphenyltin (TPT) and tributyltin (TBT) in some household products is banned by “Act on the Control of Household Products Containing Harmful Substances” in Japan. To revise the official analytical method, the method for detecting these organotin compounds was examined in six laboratories using a textile product, water-based adhesive, oil-based paint, which contained known amounts of TPT and TBT (0.1, 1.0, 10 μg/g). TPT and TBT were measured by GC-MS after ethyl-derivation with sodium tetraethylborate. The TBT recoveries in the samples were 70-120%. The TPT recoveries in the water-based adhesive samples were 80-110%, while its concentrations in the textile product and oil-based paint samples decreased because of dephenylation during storage. However, the precision of the method examined was satisfactory because most coefficients of variation for TPT and TBT in the samples were less than 10%. Furthermore, the revised method was able to detect concentrations lower than the officially regulated value. However, the sample matrix and the condition of analytical instrument might affect the estimated TPT and TBT concentrations. Therefore, the revised method may not be suitable for quantitative tests; rather, it can be employed to judge the acceptable levels of these organotin compounds by comparing the values of control sample containing regulated amounts of TPT and TBT with those for an unknown sample, with deuterated TPT and TBT as surrogate substances. It is desirable that TPT in textile and oil-based paint samples are analyzed immediately after the samples obtained because of the decomposition of TPT.