Diagnosis and molecular basis of mitochondrial respiratory chain disorders: exome sequencing for disease gene identification.

Mitochondrial disorders have the highest incidence among congenital metabolic diseases, and are thought to occur at a rate of 1 in 5000 births. About 25% of the diseases diagnosed as mitochondrial disorders in the field of pediatrics have mitochondrial DNA abnormalities, while the rest occur due to defects in genes encoded in the nucleus. The most important function of the mitochondria is biosynthesis of ATP. Mitochondrial disorders are nearly synonymous with mitochondrial respiratory chain disorder, as respiratory chain complexes serve a central role in ATP biosynthesis. By next-generation sequencing of the exome, we analyzed 104 patients with mitochondrial respiratory chain disorders. The results of analysis to date were 18 patients with novel variants in genes previously reported to be disease-causing, and 27 patients with mutations in genes suggested to be associated in some way with mitochondria, and it is likely that they are new disease-causing genes in mitochondrial disorders. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

A multifunctional envelope-type nanodevice for use in nanomedicine: concept and applications.

In the 21st century, drug development has shifted toward larger molecules such as proteins and nucleic acids, which require the use of new chemical strategies. In this process, the drug delivery system plays a central role and intracellular targeting using nanotechnology has become a key technology for the development of successful new medicines. We have developed a new delivery system, a multifunctional envelope-type nanodevice (MEND) based on "Programmed Packaging." In this new concept of packaging, multifunctional nanodevices are integrated into a nanocarrier system according to a program designed to overcome all barriers during the course of biodistribution and intracellular trafficking. In this Account, we introduce our method for delivering nucleic acids or proteins to intracellular sites of action such as the cytosol, nucleus, and mitochondria and for targeting selective tissues in vivo via systemic administration of the nanodevices. First, we introduce an octaarginine-modified MEND (R8-MEND) as an efficient intracellular delivery system, designed especially for vaccinations and transgene expression. Many types of cells can internalize the R8-MEND, mainly by inducing macropinocytosis, and the MEND escapes from macropinosomes via membrane fusion, which leads to efficient antigen presentation via the major histocompatibility complex I pathway in antigen-presenting cells. In addition, the transfection activities of the R8-MEND in dividing cells, such as HeLa or A549 cells, are as high as those for adenovirus. However, because the R8-MEND cannot induce sufficient transgene activity in primary cultured dendritic cells, which are critical regulators of the immune response, we converted the R8-MEND into a tetralamellar MEND (T-MEND). The T-MEND uses a new packaging method and delivers condensed pDNA into the nucleus via fusion between the envelopes and the nuclear membrane. To achieve efficient transfection activity, we also optimized the decondensation of nucleic acids within the nucleus. To optimize mitochondrial drug delivery, we introduced the MITOPorter. Many types of materials can be packaged into this liposome-based nanocarrier and then delivered to mitochondria via membrane fusion mechanisms. Finally, we describe an integrated strategy for in vivo tumor delivery and optimization of intracellular trafficking. Successful tumor delivery typically requires coating the surfaces of nanoparticles with PEG, but PEG can also limit uptake by the reticuloendothelial system and reduce the efficiency of intracellular trafficking. Here we integrate the optimum biodistribution and intracellular trafficking of the MEND with an innovative strategy such as enzymatically cleavable PEG and a short membrane peptide, GALA. Some of these strategies will soon be tested in the clinic.

A designed curved DNA sequence remarkably enhances transgene expression from plasmid DNA in mouse liver.

The intranuclear disposition of plasmid DNA is extremely important for transgene expression. The interactions between the plasmid DNA and the histone proteins are one of the keys for controlling the disposition. In this study, the effects of a left-handedly curved sequence (20-40 repeated A•T tracts) on transgene expression from a plasmid were examined in vivo. A naked luciferase plasmid with the curved sequence was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. Interestingly, transgene expression was markedly increased by the addition of the curved sequence. An analysis of the nucleosome positions near the left-handedly curved sequence suggested that the sequence functions as an acceptor of the histone core and allows nucleosome sliding, resulting in transcriptional activation. These results suggested that the designed curved DNA sequences could control transgene expression from plasmid DNAs in vivo.

Intracellular trafficking and transgene expression of viral and non-viral gene vectors.

The objective of this review is to summarize the critical steps in intracellular trafficking and the principal factors involved in transgene expression by a comparison of viral and non-viral gene vectors. Intracellular trafficking of viral and non-viral gene vectors are reviewed kinetically as well as from the mechanistic point of view. The peptide-dependent specific mechanism of endosomal escape by viral vectors is compared with the non-specific mechanism of non-viral vectors. Regarding the nuclear transport of DNA, a number of recently developed strategies in non-viral vectors such as the application of nuclear localization signals or cell specific transcription factors are summarized in comparison with viral nuclear gene delivery. The molecular mechanisms of transcription and the translation of delivered genes to nucleus are also summarized in view of drug delivery systems. This information is intended to serve as a basis for developing a new gene delivery system for both viral and non-viral gene vectors. Optimizing the gene delivery system by integrating this intracellular trafficking as well as transgene expression will be required in order to develop an efficient and an safe gene delivery system.

Quantitative studies on the nuclear transport of plasmid DNA and gene expression employing nonviral vectors.

The nuclear membrane is the main barrier to nonviral gene delivery. Thus, in the case of nondividing cells, a device for nuclear delivery of exogenous DNA is necessary. In addition, to precisely evaluate the efficacy of various plasmid modifications and/or nonviral vectors, it is necessary to measure, not only gene expression but also the amount of delivered plasmid DNA into the subcellular compartment, particularly the nucleus. Moreover, it is also necessary to examine effects of the state of the plasmid DNA in the nucleus or various modifications of the plasmid DNA on the process after nuclear transport, i.e., transcription. Here, we address the issues of (1) the efficient delivery of genetic materials using a nuclear localization signal (NLS), (2) the quantitative evaluation of plasmid DNA delivered to the nucleus and the relationship between the amount of plasmid DNA delivered into the nucleus and gene expression, and (3) methods for evaluating of the effect of the state of plasmid DNA on transcription in vitro.

Interactions of liposomes with cells in vitro and in vivo: opsonins and receptors.

A number of studies have appeared recently on the underlying mechanisms of liposome-cell interactions under in vitro conditions, in which isolated cell populations or cell lines were used. However, our knowledge of how liposomes interact with cells and the parameters that influence this in vivo is limited. We will summarize and discuss the relevant studies on this matter in this article. In addition, researchers in this field have long been aware of the interaction of liposomes with blood (or serum/plasma) proteins in vivo and their potential role in the process of the clearance of liposomes from the circulation. Some of the 'opsonizing' proteins, such as complement components, immunoglobulins, which enhance the interactions of liposomes with 'phagocytic cells' have been identified. However, the issue of which types of opsonins determine the fate of liposomes in vivo and how liposomal physicochemical properties such as size, charge and fluidity play an important role in the process of liposome clearance is not clear. Our own observations of one of opsonins, complement component are reviewed herein. As opposed to the fate of conventional liposomes, we briefly touch on the interaction of surface-modified liposomes, which are designed to avoid interactions with blood proteins and/or cells (sterically stabilized liposomes, long-circulating liposomes) and to actively target specific cells or tissues (targeted liposomes: immunoliposomes). Blood proteins such as opsonins are not usually thought to play an important role in the clearance of such liposomes.

Cardiomegaly in the juvenile visceral steatosis (JVS) mouse is reduced with acute elevation of heart short-chain acyl-carnitine level after L-carnitine injection.

The long-term administration of L-carnitine was very effective in preventing cardiomegaly in juvenile visceral steatosis (JVS) mice, which was confirmed by heart weight as well as the lipid contents in heart tissue. After i.p. injection of L-carnitine, the concentration of free carnitine in heart remained constant, although serum free carnitine level increased up to 80-fold. On the other hand, a significant increase in short-chain acyl-carnitine level in heart was observed. These results suggest that increased levels of short-chain acyl-carnitine, not free carnitine, might be a key compound in the protective effect of L-carnitine administration in JVS mice.

No effect of age on the dose requirement of thiopental in the rat.

With increasing human age (20-80 years), the electroencephalogram (EEG) dose requirement for the intravenous anesthetic thiopental decreases approximately 10% per decade of life. The goal of this study was to compare the dose required to attain isoelectric EEG in young (4-5 month) vs. aged (24-25-month) Fischer 344 rats. One second isoelectricity was found to be an endpoint where minimal cardiorespiratory depression occurred. The effects of age, infusion rate, and repeated administration were examined in nine young and nine old rodents. Thiopental dose requirement increased with increasing infusion rates. Repeated administration at two-day intervals did not demonstrate tolerance to thiopental. No difference in thiopental dose requirement was detected in the young vs. elderly rats. In a separate group of five young and five old rats, thiopental plasma, brain, heart, and CSF concentrations were measured when five seconds of EEG isoelectricity was achieved: no consistent differences were noted. The rat may not be an appropriate model to investigate acute age-related anesthetic effects in humans, because cardiovascular changes with age are dissimilar between species.

3-D computer graphics based on integral photography.

Integral photography (IP), which is one of the ideal 3-D photographic technologies, can be regarded as a method of capturing and displaying light rays passing through a plane. The NHK Science and Technical Research Laboratories have developed a real-time IP system using an HDTV camera and an optical fiber array. In this paper, the authors propose a method of synthesizing arbitrary views from IP images captured by the HDTV camera. This is a kind of image-based rendering system, founded on the 4-D data space Representation of light rays. Experimental results show the potential to improve the quality of images rendered by computer graphics techniques.

Development of a pharmacokinetic/pharmacodynamic (PK/PD)-simulation system for doxorubicin in long circulating liposomes in mice using peritoneal P388.

The objective of this study was to develop a simulation system that optimizes the pharmacokinetic parameters of drug carriers for anticancer agents in order to maximize their anticancer effects. The pharmacokinetic/pharmacodynamic (PK/PD) model of doxorubicin (DOX) encapsulated into liposomes has been developed for mice and each parameter required for simulations was obtained in the peritoneally inoculated P388 leukemia model in mice. PK parameters, which describe the dispositions of free and liposomally encapsulated DOX, were obtained by kinetic analysis of experimental data in this study, as well as from literature. PD parameters, which describe the growth and death rate of cancer cells in vivo, were also determined. The PK/PD model developed in this study is capable of simulating the time course of the number of cancer cells quantitatively and evaluating the significance of each parameter on the carrier system for DOX. Simulations based on the PK/PD model predict the optimum rate of drug release from long circulating liposomes as 0.06 h(-1) for maximum anticancer effect. Thus, this simulation system provides useful information relative to the optimization of drug carriers for DOX.

Optimization of antitumor effect of liposomally encapsulated doxorubicin based on simulations by pharmacokinetic/pharmacodynamic modeling.

It has been reported that long circulating liposomes enhanced the antitumor effect of doxorubicin (DOX) by increasing delivery of DOX to tumor tissues. However, there is no quantitative information on the relationship between the antitumor effect and liposomal characteristics governing the release rate of entrapped drugs, although the importance of drug release-rate control from liposomes has been pointed out. Here, we developed a physiological model for free and liposomal DOX to calculate the time course of free DOX in the extracellular space and linked this with a cell kill kinetic model to quantify the antitumor effect of DOX. Simulations were performed to clarify the relationship between antitumor effect and pharmacokinetic or physicochemical parameters of liposomes, as well as pharmacological or physiological parameters of tumor tissues. The importance of long circulation time of liposomes was confirmed. The optimum rate of drug release from long circulating liposomes was found at the release rate constant of around 0.06 h(-1). This optimum value was not dependent on the tumor proliferation time, sensitivity of tumor cells to DOX, or the tumor blood flow-rate. This simulation indicated that the optimization of the delivery to tumor tissue by long circulating liposomes could be possible by changing the release rate of DOX for the maximum antitumor effect.

The complement- but not mannose receptor-mediated phagocytosis is involved in the hepatic uptake of cetylmannoside-modified liposomes in situ.

In the elimination of injected liposomes in vivo, it is considered that several serum components play an important role on hepatic uptake of them. This study was conducted to clarify the hepatic uptake mechanism of cetylmannoside (Man)-modified multilamellar vesicles (Man-MLV) using perfused rat liver. In the presence of serum, Man-MLV was taken up by the liver depending on the serum concentration, and it showed an approximately two-fold higher accumulation than MLV without any surface modifications (PC-MLV). These hepatic uptakes of liposomes were obviously inhibited by preheating the serum at 56 degrees C for thirty minutes or by the treatment with anti-rat C3 antiserum. Further, SDS-PAGE followed by immunoblot analysis showed the deposition of iC3b on the opsonized Man-MLV. These results obtained in the present study suggested that hepatic uptake of Man-MLV was mainly mediated by complement receptor rather than mannose receptor on Kupffer cells in vivo.

Size dependent liposome degradation in blood: in vivo/in vitro correlation by kinetic modeling.

The degradation of liposomes in blood circulation is important in regulating the releasing rate of encapsulated compounds. In this study, the effect of liposome size--one of the principal determining factors in liposome disposition--on their degradation in serum/blood was evaluated quantitatively both in vitro and in vivo. In the in vitro study, the time courses of the degradation of liposomes in fresh rat serum were measured continuously using 5(6)-carboxyfluorescein (CF) as an aqueous phase marker and were described by the kinetic model with the lag time (tau), first order degradation rate constant (k), and the maximum degradation (alpha). Both k and alpha increased with the increase of liposome size, which indicated a higher affinity of larger liposomes for complement activation. In the in vivo study, the degradation of liposomes was evaluated sensitively by a first order degradation rate constant (kd) in blood circulation. The kd was obtained by kinetically modeling the liposome degradation in vivo using 3H-inulin as an aqueous phase marker. The size dependent kd correlated well with the hepatic uptake clearance, which suggests an underlying complement activation mechanism common to both degradation and hepatic uptake of liposomes. There was a good correlation in the degradation rate constant between in vitro and in vivo trials. These kinetic analyses validate the quantitative evaluation of liposome degradation in blood circulation and provide a useful way to predict the degradation of liposomes in vivo from in vitro experiments.

Separation of fine crackles from vesicular sounds by a nonlinear digital filter.

Crackles are discontinuous adventitious sounds, and their separation is an important process in the analysis of lung sounds. In order to separate the crackles from vesicular sounds automatically, we used a nonlinear digital filter which was designed to separate nonstationary from stationary signals. We applied this filter to the lung sounds recorded from six patients with pulmonary fibrosis. The separation was satisfactory enough to make this method useful in clinical medicine.

Intracellular control of gene trafficking using liposomes as drug carriers.

The objective of this review is to summarize some of the critical barriers in gene delivery and recent progress in overcoming such barriers using non-viral carrier systems. Receptor-mediated endocytosis is generally considered to be a principal entering pathway. Therefore, endosomal escape is an essential step for achieving efficient transfection. The nuclear membrane is also a critical barrier in gene delivery and the application of the nuclear localization signal is discussed, based on recent strategies. It is essential to optimize the carrier system, in order to enhance the transfection ability equivalent to a viral system. The importance of developing an intracellular pharmacokinetic model of genes is emphasized in the optimization of non-viral carrier systems.

Biodistribution of liposomes and C3 fragments associated with liposomes: evaluation of their relationship.

The biodistribution of liposomes with two different kind phospholipids (hydrogenated egg phosphatidylcholine and egg phosphatidylcholine) plus cholesterol (CHOL) were investigated after intravenous administration to rats. Elimination of liposomes from blood circulation was affected by the lipid composition. It appeared that the inclusion of CHOL in liposomes accelerates the rate of liposome uptake by liver, resulting in rapid elimination of liposomes. The amount of C3 fragments bound to liposomes was quantitatively determined to assess the contribution of the complement system to liposome accumulation into organs and liposome destabilization in vivo and in vitro. The amount of bound C3 fragments was directly proportional to CHOL content, and the amount was also proportional to the CLh, CLs as well as CLrel. This relationship suggests that the complement system is responsible for the elimination of liposomes from blood circulation, presumably as a consequence of opsonization by C3 fragments and assembly of membrane attack complex (MAC) onto liposomes. In addition, substitution of cholesteryl methyl ether into the liposome formulation for CHOL significantly diminished not only the binding of C3 fragments but also the CLh, CLs and CLrel, resulting in increased mean resident time (MRT) of the liposomes. This result suggests that the hydroxyl-group on CHOL is a binding site for C3 fragments on the liposomes and that CHOL in a liposome formulation promotes the accumulation of liposomes into the liver and spleen, probably due to their uptake by phagocytic cells, and impairs the stability of the liposomes in blood circulation, via a mechanism involving the complement system.

The complement system enhances the clearance of phosphatidylserine (PS)-liposomes in rat and guinea pig.

In this study, we investigated the contribution of the complement system to the biodistribution of phosphatidylserine (PS)-containing liposomes in rat and guinea pig. It appeared that the inclusion of PS in the liposome formulation accelerates the rate of liposome uptake by liver, resulting in rapid elimination of the liposomes from blood circulation. Pretreatment with K76COOH (K76), an anti-complement agent, decreased the rapid uptake of PS-containing liposomes by guinea pig liver, resulting in increasing blood concentration of the liposomes. Significant complement-dependent liposome destabilization was observed in vitro in both animals, whereas the complement-dependent destabilization in vivo was likely only a part of the process of the clearance of the PS-containing liposomes. This discrepancy suggests that the rate of complement-dependent liposome uptake by liver is much faster than the rate of complement-dependent liposome destabilization in vivo. Pretreatment of K76 dramatically inhibited the binding of C3 fragments, one of dominant opsonins, to PS-containing liposomes in guinea pig under both in vivo and in vitro conditions. This finding suggests that the C3 fragments in the system are responsible for the clearance of the PS-containing liposomes in guinea pig. In rat, in contrast to guinea pig, in vivo binding of C3 fragments was not inhibited by K76-pretreatment, while in vitro binding was inhibited. This discrepancy may be due to different experimental conditions between in vitro and in vivo assay. Nevertheless, based on the observations in this study, the complement components are most likely involved in the clearance of the PS-containing liposomes in rat. Taken together, the activity of PS in enhancing the liposome clearance appears to be mediated by the complement components, presumably C3 fragments, in both guinea pig and rat. This is a first report showing the mechanism on the hepatic uptake of the PS-containing liposomes in guinea pig.

Effect of cholesterol content in activation of the classical versus the alternative pathway of rat complement system induced by hydrogenated egg phosphatidylcholine-based liposomes.

Liposomes composed of hydrogenated egg phosphatidylcholine (HEPC) and cholesterol (CHOL) were found to activate the rat complement (C) system in a CHOL content-dependent manner. Liposomes containing 22 or 33 mol% CHOL activated the C system in a Ca(2+)-dependent manner, suggesting that C activation occurred via the classical pathway. Liposomes containing 44 mol% CHOL activated the C system in a Ca(2+) independent manner, suggesting that C activation occurred via the alternative pathway. The CHOL content appeared to dictate the pathway by which the C system was activated. This C activation was inhibited by removal of serum component(s), which adsorb to the liposomes. Activation of the alternative pathway, induced by the liposomes, was reduced by the depletion of IgG and IgM, whereas the classical pathway activation was reduced by the depletion of IgG, but not IgM. In addition, the removal of adsorbed serum component(s) by treatment with 44 mol% CHOL-containing liposomes decreased serum IgG and IgM levels that adsorb to the same liposomes, whereas the removal of adsorbed serum component(s) by treatment with 22 mol% CHOL-containing liposomes only slightly decreased serum IgG levels, which adsorbs to the same liposomes. Collectively, both IgG and IgM, which are specifically adsorbed to the liposomes in a CHOL-content dependent manner, were responsible for C activation via the alternative pathway induced by the 44 mol% CHOL containing liposomes. IgG alone would be partially responsible for C activation via the classical pathway induced by 22 or 33 mol% CHOL-containing liposomes. The discovery of this unique C-activating property of liposomes will be of value in attempts to decipher the underlying mechanism of C activation by providing a useful model membrane system.

Saturable, non-Michaelis-Menten uptake of liposomes by the reticuloendothelial system.

Multilamellar vesicles (300-350 nm) were infused into the rat femoral vein at the rate of 4, 40 and 400 nmol phosphatidycholine min-1 for 6 h using [3H]inulin as an aqueous marker. The time courses of blood concentration of vesicles, normalized for infusion rate, were not superimposable, showing the non-linearity of liposome disposition in the blood circulation. These time courses of blood concentration were well fitted by a single Michaelis-Menten equation. On the other hand, the time courses of tissue content could not be so accommodated. Additionally, the observed relationship between the uptake of liposomes by the liver and their clearance from it and other organs differed essentially from a simulation based on Michaelis-Menten type saturable kinetics. Therefore, it is suggested that there is a time-dependent non-Michaelis-Menten type process in the phagocytosis of macrophages in the reticuloendothelial system.