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The cytotoxic mycotoxin deoxynivalenol (DON) reportedly has adverse effects on oocyte maturation and embryonic development in pigs. Recently, the interplay between cell apoptosis and endoplasmic reticulum (ER) stress has garnered increasing attention in embryogenesis. However, the involvement of the inositol-requiring enzyme 1 (IRE1)/c-jun N-terminal kinase (JNK)/C/EBP homologous protein (CHOP) pathways of unfolded protein response (UPR) signaling in DON-induced apoptosis in porcine embryos remains unknown. In this study, we revealed that exposure to DON (0.25 μM) substantially decreased cell viability until the blastocyst stage in porcine embryos, concomitant with initiation of cell apoptosis through the IRE1/JNK/CHOP pathways in response to ER stress. Quantitative PCR confirmed that UPR signaling-related transcription factors were upregulated in DON-treated porcine blastocysts. Western blot analysis showed that IRE1/JNK/CHOP signaling was activated in DON-exposed porcine embryos, indicating that ER stress-associated apoptosis was instigated. The ER stress inhibitor tauroursodeoxycholic acid protected against DON-induced ER stress in porcine embryos, indicating that the toxic effects of DON on early developmental competence of porcine embryos can be prevented. In conclusion, DON exposure impairs the developmental ability of porcine embryos by inducing ER stress-mediated apoptosis via IRE1/JNK/CHOP signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The treatment of chronic Achilles tendonitis (AT) often requires prolonged therapy and invasive therapeutic methods such as surgery or therapeutic endoscopy. To prevent the progression of chronic AT, excessive inflammation must be alleviated at an early stage. Corticosteroids or nonsteroidal anti-inflammatory drugs are generally prescribed to control inflammation; however, the high doses and long therapeutic periods required may lead to serious side effects. Herein, a local injectable poly(organophosphazene) (PPZ) - celecoxib (CXB) nanoparticle (PCNP) hydrogel system with long-term anti-inflammatory effects was developed for the treatment of tendonitis. The amphiphilic structure and thermosensitive mechanical properties of PPZ means that the hydrophobic CXB can be easily incorporated into the hydrophobic core to form PCNP at 4 °C. Following the injection of PCNP into the AT, PCNP hydrogel formed at body temperature and induced long-term local anti-inflammatory effects via sustained release of the PCNP. The therapeutic effects of the injectable PCNP system can alleviate excessive inflammation during the early stages of tissue damage and boost tissue regeneration. This study suggests that PCNP has significant potential as a long-term anti-inflammatory agent through sustained nonsteroidal anti-inflammatory drugs (NSAIDs) delivery and tissue regeneration boosting. STATEMENT OF SIGNIFICANCE: In the treatment of Achilles tendinitis, a long-term anti-inflammatory effect is needed to alleviate excessive inflammation and induce regeneration of the damaged Achilles tendon. Injectable poly(organophosphazene)(PPZ)-celecoxib(CXB) nanoparticles (PCNP) generated a long-term, localized-anti-inflammatory effect in the injected region, which successfully induced the expression of anti-inflammatory cytokines and suppressed pro-inflammatory cytokines, while the PCNPs degraded completely. Accordingly, regeneration of the damaged Achilles tendon was achieved through the long-term anti-inflammatory effect induced by a single PCNP injection. The PCNP system therefore has great potential in long-term NSAIDs delivery for various tissue engineering applications., Competing Interests: Declaration of Competing Interest The authors declare no competing interests, (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Treatment of osteoarthritis (OA) by administration of corticosteroids is a commonly used method in clinics using anti-inflammatory medicine. Oral administration or intra-articular injection of corticosteroids can reduce the pain and progress of cartilage degeneration, but they are usually insufficient to show local and long-term anti-inflammatory effects because of their fast clearance in the body. In this study, we suggest an injectable anti-OA drug depot system for sustained drug release that provides long-term effective therapeutic advantages. Amphiphilic poly(organophosphazene), which has temperature-dependent nanoparticle forming and sol-gel transition behaviors when dissolved in aqueous solution, was synthesized for triamcinolone acetonide (TCA) delivery. Because hydrophobic parts of the polymer can interact with hydrophobic parts of the TCA, the TCA was encapsulated into the self-assembled polymeric nanoparticles. The TCA-encapsulated polymeric nanoparticles (TePNs) were well dispersed in an aqueous solution below room temperature so that they can be easily injected as a sol state into an intra-articular region. However, the TePNs solution transforms immediately to a viscose 3D hydrogel like a synovial fluid in the intra-articular region via the conducted body temperature. An in vitro TCA release study showed sustained TCA release for six weeks. One-time injection of the TePN hydrogel system in an early stage of OA-induced rat model showed a great inhibition effect against further OA progression. The OA-induced knees completely recovered as a healthy cartilage without any abnormal symptoms., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

In this study we investigated the contact characteristics of human prostate cancer cells (PC3) on silicon micropillar arrays with complex shapes by using high-resolution confocal fluorescence microscopy techniques. These arrays consist of micropillars that are of various cross-sectional geometries which produce different deformation profiles in adherent cells. Fluorescence micrographs reveal that some DAPI (4',6-diamidino-2-phenylindole)-stained nuclei from cells attached to the pillars develop nanometer scale slits and contain low concentrations of DNA. The lengths of these slits, and their frequency of occurrence, were characterized for various cross-sectional geometries. These DNA-depleted features are only observed in locations below the pillar's top surfaces. Results produced in this study indicate that surface topography can induce unique nanometer scale features in the PC3 cell., Competing Interests: The authors declare no conflicts of interest.

For a substance to be used as a drug delivery carrier and tissue inducible material for a target disease, its drug release rate and physical properties should be optimized to facilitate the healing process. We developed multi-tunable hydrogel systems with various physical properties and release behaviors to determine the optimal conditions for bone regeneration. Five injectable poly(phosphazene) hydrogels were developed with different types and amounts of anionic side-chains. The five polymer hydrogels showed considerably different in vitro and in vivo performances for sol-gel phase transition, dissolution/degradation, water uptake, and pore size. Furthermore, bone morphogenetic protein-2 (BMP-2) was loaded into the polymer hydrogels by forming nano-sized ionic-complexes with each polymer. The five types of nanocomplex hydrogels showed completely different BMP-2 release rates. By administering each nanocomplex hydrogel to mouse calvarial, we identified the most adapted nanocomplex hydrogel system for effective bone regeneration. The BMP-2 release rate was the most important factor in effective bone regeneration. Finally, the bone regeneration effect of the optimized hydrogel system was investigated in a critical-sized calvarial defect model., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: In the present study, various freezing containers were tested for mouse embryos of respective developmental stages; embryos were vitrified and then their survival rate and developmental rate were monitored. Mouse two cell, 8 cell, and blastula stage embryos underwent vitrification freezing-thawing and then their recovery rate, survival rate, development rate, and hatching rate were investigated., Methods: EM-grid, OPS, and cryo-loop were utilized for vitrification freezing-thawing of mouse embryos., Results: It was found that recovery rate and survival rate were higher in the group of cryo-loop compared to those of EM-grid (p < 0.05). Embryonic development rate, two cell embryos to blastocyst, as well as hatching rate were higher in the control group compared to the EM-grid group and OPS group (p < 0.05), yet no difference was noted between the control group and cryo-loop group. Development rate and hatching rate of eight cell morulae and blastocysts were all lower in the treatment groups than the control group whilst hatching rate of blastocysts was higher in the control group compared to the groups of EM-grid and OPS (p < 0.05); although the cryo-loop group was shown to be slightly higher than other groups, it was not statistically significant., Conclusions: In the study, we investigate effects of freezing containers on vitrified embryos of respective developmental stages; it was demonstrated that higher developmental rate was shown in more progressed (or developed) embryos with more blastomeres. There was however, no difference in embryonic development rate was shown amongst containers. Taken together, further additional studies are warranted with regards to 1) manipulation techniques of embryos for various vitrification freezing containers and 2) preventive measures against contamination via liquid nitrogen.

Background: This study was investigated the effects of culture media conditions on production of eggs fertilized in vitro of embryos from ovaries of high grade Korean native cow, Hanwoo., Methods: The IVMD 101 and IVF 100 were used for in vitro maturation of selected Hanwoo oocytes and In vitro embryo culture after in vitro fertilization, respectively. The IVMD 101 and IVD 101 were used for in vitro culture and completely free of serum., Results: The cleavage rates of 2-cell embryos in reference to Hanwoo oocytes were 86.7, 92.9 , and 90.1 % in the control group, IVDM101 medium and IVD101 medium, respectively which indicates that the IVDM101 medium and IVD101 medium may result favorable outcomes. The in vitro development rates of blastocysts were 12.4, 38.4 and 32.4 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. For hatched blastocysts, it was 5.3, 33.9, and 28.6 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. Hence, more favorable results were expected for the hatched blastocysts in which the IVMD101 medium and IVD101 medium were used than the control group. Average cell numbers of blastocysts were 128.3, 165.7, and 163.6 in the groups of TCM-199 + 10 % FBS medium, IVMD 101 medium, and IVD 101 medium, respectively which clearly show that the IVMD 101 and IVD 101 medium consequence significantly higher cell numbers compared to the control group (i.e., TCM-199 + 10 % FBS medium). Pregnancy rate after embryo transfer was 39.6 % when the serum free medium was used which is higher than that of the medium supplemented with serum (32.8 %). In addition, stillbirth rates were 4.9 % in the group of serum free medium whereas it was 13.6 % in the serum supplemented medium (13.6 %)., Conclusions: Taken altogether, serum free media, the IVMD 101 and IVD 101 represented more favorable results in the embryo development rate of embryos, cell numbers of blastocyst, and pregnancy rate. Of note, the IVMD 101 medium showed better outcomes hence, it might be a better option for future applications for in vitro culture of bovine embryos.

Metallic nanopillar/nanowires are emerging as promising platforms for biological applications, as they allow for the direct characterization and regulation of cell function. Herein we study the response of cells to a versatile nanopillar platform. Nanopillar arrays of various shape, size, and spacing and different nanopillar-substrate interfacial strengths were fabricated and interfaced with fibroblasts and several unique cell-nanopillar interactions were observed using high resolution scanning electron microscopy. Nanopillar penetration, engulfment, tilting, lift off and membrane thinning, were observed by manipulating nanopillar material, size, shape and spacing. These unique cell responses to various nanostructures can be employed for a wide range of applications including the design of highly sensitive nano-electrodes for single-cell probing., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: In search of an ideal method of assisted hatching (AH), we compared the effects of conventional micropipette-AH and laser-AH on the blastocyst formation rate (BFR) and blastocyst cell numbers., Methods: Four- to five-week-old ICR female mice were paired with male mice after superovulation using Pregnant mare's serum gonadotropin (PMSG) and hCG. The two-cell embryos were flushed from the oviducts of female mice. The retrieved two-cell embryos underwent one of five AH procedures: single mechanical assisted hatching (sMAH); cross mechanical assisted hatching (cMAH); single laser assisted hatching (sLAH); quarter laser assisted hatching (qLAH); and quarter laser zona thinning assisted hatching (qLZT-AH). After 72 hours incubation, double immunofluorescence staining was performed., Results: Following a 72 hours incubation, a higher hatching BFR was observed in the control, sMAH, cMAH, and sLAH groups, compared to those in the qLAH and qLZT-AH groups (p<0.05). The hatched BFR was significantly higher in the qLAH and qLZT-AH groups than in the others (p<0.05 for each group). The inner cell mass (ICM) was higher in the control and sMAH group (p<0.05). The trophectoderm cell number was higher in the cMAH and qLAH groups (p<0.05)., Conclusion: Our results showed that the hatched BFR was higher in groups exposed the the qLAH and qLZT-AH methods compared to groups exposed to other AH methods. In the qLAH group, although the total cell number was significantly higher than in controls, the ICM ratio was significantly lower in than controls.

A broad range of human diseases are associated with bacterial infections, often initiated by specific adhesion of a bacterium to the target environment. Despite the significant role of bacterial adhesion in human infectious diseases, details and mechanisms of bacterial adhesion have remained elusive. Herein, we study the physical interactions between Staphylococcus aureus, a type of micro-organism relevant to infections associated with medical implants, and nanocrystalline (nc) nickel nanostructures with various columnar features, including solid core, hollow, x-shaped and c-shaped pillars. Scanning electron microscopy results show the tendency of these bacterial cells to attach to the nickel nanostructures. Moreover, unique single bacterium attachment characteristics were observed on nickel nanostructures with dimensions comparable to the size of a single bacterium., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Despite advances in clinical therapy, metastasis remains the leading cause of death in breast cancer patients. Mutations in mitochondrial DNA, including those affecting complex I and oxidative phosphorylation, are found in breast tumors and could facilitate metastasis. This study identifies mitochondrial complex I as critical for defining an aggressive phenotype in breast cancer cells. Specific enhancement of mitochondrial complex I activity inhibited tumor growth and metastasis through regulation of the tumor cell NAD+/NADH redox balance, mTORC1 activity, and autophagy. Conversely, nonlethal reduction of NAD+ levels by interfering with nicotinamide phosphoribosyltransferase expression rendered tumor cells more aggressive and increased metastasis. The results translate into a new therapeutic strategy: enhancement of the NAD+/NADH balance through treatment with NAD+ precursors inhibited metastasis in xenograft models, increased animal survival, and strongly interfered with oncogene-driven breast cancer progression in the MMTV-PyMT mouse model. Thus, aberration in mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, while therapeutic normalization of the NAD+/NADH balance can inhibit metastasis and prevent disease progression.

A dual ionic interaction system composed of a positively charged polyelectrolyte complex (PEC) containing human growth hormone (hGH) and anionic thermosensitive hydrogel has been suggested for sustained delivery of bioactive hGH. The PEC was prepared by ionic interaction between negatively charged hGH and positively charged protamine sulfate (PS) to suppress diffusion of hGH. Moreover, we loaded the positively charged PEC into an anionic, injectable, and thermosensitive poly(organophosphazene) hydrogel to enhance sustained release of hGH by dual ionic interactions. PS formed a spherical complex with hGH, and their ionic interaction grew stronger with increasing amounts of PS. From a weight ratio of 0.5, the PS/hGH complex had a size and zeta-potential that were constantly maintained around 500 nm and +8 mV, respectively, in 0.9% NaCl. The PEC-loaded hydrogels suppressed the initial burst release of hGH and extended the release period in vitro and in vivo. In a pharmacokinetic study in rats, the PEC-loaded anionic hydrogel extended half-life 13-fold with similar area under the curve (AUC) compared to hGH solution. Furthermore, single injection of PEC-loaded anionic hydrogel showed a more increased growth rate than daily injection of hGH solution for 7 days in hypophysectomized rats, demonstrating its potential as an injectable, sustained delivery system that can release bioactive hGH., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We have endeavored to develop injectable, thermosensitive, biodegradable hydrogels that prolong human growth hormone (hGH) release, improving bioavailability through introducing balanced ionic interactions. Cationic poly(organophosphazene)-polyethylenimine (PEI, 1.8 kDa) conjugate hydrogels were synthesized as those hydrogels for sustained delivery of anionic hGH with proper ionic strength of association/dissociation. We have additionally prepared different chain lengths of α-amino-ω-methoxy-poly(ethylene glycol) (AMPEG550 and AMPEG750) for the synthesis of conjugates as a means to control hydrogel degradation rates. All Aqueous solutions of PEI-conjugates became hydrogels hydrolyzable in proportion to AMPEG molecular weight at body temperature; these PEI-conjugates complexed with hGH and extended hGH release in vitro. In pharmacokinetic studies of hGH behavior in SD rats, hydrogels of PEI-conjugate/hGH complexes could suppress the initial burst-phase, and extend the duration, of release, as well as increasing of area under the curve (AUC) compared to controls including hGH solution or non-ionic hydrogel. In a hypophysectomized rat model, the biological efficacy of hGH delivered from PEI-conjugate/hGH complex hydrogels was equivalent to that from daily administration over four days based on body weight gain and width of the tibial growth plate. These results suggest that ionic, thermosensitive, poly(organophosphazene)-PEI-conjugate hydrogel demonstrates potential as an injectable depot for sustained delivery of bioavailable hGH., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Gangliosides have been suggested to play important roles in various functions such as adhesion, cell differentiation, growth control, and signaling. Mouse follicular development, ovulation, and luteinization during the estrous cycle are regulated by several hormones and cell-cell interactions. In addition, spermatogenesis in seminiferous tubules of adult testes is also regulated by several hormones, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and cell-cell interactions. The regulation of these processes by hormones and cell-cell interactions provides evidence for the importance of surface membrane components, including gangliosides. During preimplantation embryo development, a mammalian embryo undergoes a series of cleavage divisions whereby a zygote is converted into a blastocyst that is sufficiently competent to be implanted in the ma ternal uterus and continue its development. Mouse embryonic stem (mES) cells are pluripotent cells derived from mouse embryo, specifically, from the inner cell mass of blastocysts. Differentiated neuronal cells are derived from mES cells through the formation of embryonic bodies (EBs). EBs recapitulate many aspects of lineage-specific differentiation and temporal and spatial gene expression patterns during early embryogenesis. Previous studies on ganglioside expression during mouse embryonic development (including during in vitro fertilization, ovulation, spermatogenesis, and embryogenesis) reported that gangliosides were expressed in both undifferentiated and differentiated (or differentiating) mES cells. In this review, we summarize some of the advances in our understanding of the functional roles of gangliosides during the stages of mouse embryonic development, including ovulation, spermatogenesis, and embryogenesis, focusing on undifferentiated and differentiated mES cells (neuronal cells).

Background: The rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi1, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. In the so-called transkingdom gene therapy, one major concern is the fact that the yeast protein is foreign in mammals. Long term expression of Ndi1 observed in rodents with no apparent damage to the target tissue was indicative of no action by the host's immune system., Methodology/principal Findings: In the present study, we examined rat skeletal muscles expressing Ndi1 for possible signs of inflammatory or immune response. In parallel, we carried out delivery of the GFP gene using the same viral vector that was used for the NDI1 gene. The tissues were subjected to H&E staining and immunohistochemical analyses using antibodies specific for markers, CD11b, CD3, CD4, and CD8. The data showed no detectable signs of an immune response with the tissues expressing Ndi1. In contrast, mild but distinctive positive reactions were observed in the tissues expressing GFP. This clear difference most likely comes from the difference in the location of the expressed protein. Ndi1 was localized to the mitochondria whereas GFP was in the cytosol., Conclusions/significance: We demonstrated that Ndi1 expression did not trigger any inflammatory or immune response in rats. These results push forward the Ndi1-based molecular therapy and also expand the possibility of using foreign proteins that are directed to subcellular organelle such as mitochondria.

Defects in mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) have been implicated in a number of acquired and hereditary diseases including Leigh's syndrome and more recently Parkinson's disease. A limited number of strategies have been attempted to repair the damaged complex I with little or no success. We have recently shown that the non-proton-pumping, internal NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae (baker's yeast) can be successfully inserted into the mitochondria of mice and rats, and the enzyme was found to be fully active. Using recombinant adenoassociated virus vectors (serotype 5) carrying our NDI1 gene, we were able to express the Ndi1 protein in the substantia nigra (SN) of C57BL/6 mice with an expression period of two months. The results show that the AAV serotype 5 was highly efficient in expressing Ndi1 in the SN, when compared to a previous model using serotype 2, which led to nearly 100% protection when using an acute MPTP model. It is conceivable that the AAV-serotype5 carrying the NDI1 gene is a powerful tool for proof-of-concept study to demonstrate complex I defects as the causable factor in diseases of the brain.

Background: Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder with point mutations in mitochondrial DNA which result in loss of vision in young adults. The majority of mutations reported to date are within the genes encoding the subunits of the mitochondrial NADH-quinone oxidoreductase, complex I. Establishment of animal models of LHON should help elucidate mechanism of the disease and could be utilized for possible development of therapeutic strategies., Methodology/principal Findings: We established a rat model which involves injection of rotenone-loaded microspheres into the optic layer of the rat superior colliculus. The animals exhibited the most common features of LHON. Visual loss was observed within 2 weeks of rotenone administration with no apparent effect on retinal ganglion cells. Death of retinal ganglion cells occurred at a later stage. Using our rat model, we investigated the effect of the yeast alternative NADH dehydrogenase, Ndi1. We were able to achieve efficient expression of the Ndi1 protein in the mitochondria of all regions of retinal ganglion cells and axons by delivering the NDI1 gene into the optical layer of the superior colliculus. Remarkably, even after the vision of the rats was severely impaired, treatment of the animals with the NDI1 gene led to a complete restoration of the vision to the normal level. Control groups that received either empty vector or the GFP gene had no effects., Conclusions/significance: The present study reports successful manifestation of LHON-like symptoms in rats and demonstrates the potential of the NDI1 gene therapy on mitochondrial optic neuropathies. Our results indicate a window of opportunity for the gene therapy to be applied successfully after the onset of the disease symptoms.

It is widely recognized that mitochondrial dysfunction, most notably defects in the NADH-quinone oxidoreductase (complex I), is closely related to the etiology of sporadic Parkinson's disease (PD). In fact, rotenone, a complex I inhibitor, has been used for establishing PD models both in vitro and in vivo. A rat model with chronic rotenone exposure seems to reproduce pathophysiological conditions of PD more closely than acute mouse models as manifested by neuronal cell death in the substantia nigra and Lewy body-like cytosolic aggregations. Using the rotenone rat model, we investigated the protective effects of alternative NADH dehydrogenase (Ndi1) which we previously demonstrated to act as a replacement for complex I both in vitro and in vivo. A single, unilateral injection of recombinant adeno-associated virus carrying the NDI1 gene into the vicinity of the substantia nigra resulted in expression of the Ndi1 protein in the entire substantia nigra of that side. It was clear that the introduction of the Ndi1 protein in the substantia nigra rendered resistance to the deleterious effects caused by rotenone exposure as assessed by the levels of tyrosine hydroxylase and dopamine. The presence of the Ndi1 protein also prevented cell death and oxidative damage to DNA in dopaminergic neurons observed in rotenone-treated rats. Unilateral protection also led to uni-directional rotation of the rotenone-exposed rats in the behavioral test. The present study shows, for the first time, the powerful neuroprotective effect offered by the Ndi1 enzyme in a rotenone rat model of PD.

Defects in the proton-translocating NADH-quinone oxidoreductase (complex I) of mammalian mitochondria are linked to neurodegenerative disorders. The mechanism leading to cell death elicited by complex I deficiency remains elusive. We have shown that expression of a rotenone-insensitive yeast NADH-quinone oxidoreductase (Ndi1) can rescue mammalian cells from complex I dysfunction. By using the Ndi1 enzyme, we have investigated the key events in the process of cell death using a rat dopaminergic cell line, PC12. We found that complex I inhibition provokes the following events: 1) activation of specific kinase pathways; 2) release of mitochondrial proapoptotic factors, apoptosis inducing factor, and endonuclease G. AS601245, a kinase inhibitor, exhibited significant protection against these apoptotic events. The traditional caspase pathway does not seems to be involved because caspase 3 activation was not observed. Our data suggest that overproduction of reactive oxygen species (ROS) caused by complex I inhibition is responsible for triggering the kinase activation, for the release of the proapoptotic factors, and then for cell death. Nearly perfect prevention of apoptotic cell death by Ndi1 agrees with our earlier observation that the presence of Ndi1 diminishes rotenone-induced ROS generation from complex I. In fact, this study demonstrated that Ndi1 keeps the redox potential high even in the presence of rotenone. Under these conditions, ROS formation by complex I is known to be minimal. Possible use of our cellular model is discussed with regard to development of therapeutic strategies for neurodegenerative diseases caused by complex I defects.

Using rat dopaminergic and human neuroblastoma cell lines transduced with the NDI1 gene encoding the internal NADH dehydrogenase (Ndi1) from Saccharomyces cerevisiae, we investigated reactive oxygen species (ROS) generation caused by complex I inhibition. Incubation of non-transduced cells with rotenone elicited oxidative damage to mitochondrial DNA as well as lipid peroxidation. In contrast, oxidative stress was significantly decreased when the cells were transduced with NDI1. Furthermore, mitochondria from the NDI1-transduced cells showed a suppressed rate of ROS formation by the complex I inhibitors. We conclude that the Ndi1 enzyme is able to suppress ROS overproduction from defective complex I.

Recent studies suggest that dysfunction of the NADH-quinone oxidoreductase (complex I) is associated with a number of human diseases, including neurodegenerative disorders such as Parkinson disease. We have shown previously that the single subunit rotenone-insensitive NADH-quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae mitochondria can restore NADH oxidation in complex I-deficient mammalian cells. The Ndi1 enzyme is insensitive to complex I inhibitors such as rotenone and 1-methyl-4-phenylpyridinium ion, known as a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To test the possible use of the NDI1 gene as a therapeutic agent in vivo, we chose a mouse model of Parkinson disease. The NDI1-recombinant adeno-associated virus particles (rAAV-NDI1) were injected unilaterally into the substantia nigra of mice. The animals were then subjected to treatment with MPTP. The degree of neurodegeneration in the nigrostriatal system was assessed immunohistochemically through the analysis of tyrosine hydroxylase and glial fibrillary acidic protein. It was evident that the substantia nigra neurons on the side used for injection of rAAV-NDI1 retained a high level of tyrosine hydroxylase-positive cells, and the ipsilateral striatum exhibited significantly less denervation than the contralateral striatum. Furthermore, striatal concentrations of dopamine and its metabolites in the hemisphere that received rAAV-NDI1 were substantially higher than those of the untreated hemisphere, reaching more than 50% of the normal levels. These results indicate that the expressed Ndi1 protein elicits resistance to MPTP-induced neuronal injury. The present study is the first successful demonstration of complementation of complex I by the Ndi1 enzyme in animals.

Neuronal damage subsequent to transient cerebral ischemia is a multifactorial process involving several overlapping mechanisms. Gangliosides, sialic acid-conjugated glycosphingolipids, reduce the severity of acute brain damage in vitro. However their in vivo effects on the cerebral cortex damaged by ischemic infarct are unknown. To assess the possible protective role of gangliosides we examined their expression in the cerebral cortex damaged by ischemic infarct in the rat. Ischemia was induced by middle cerebral artery (MCA) occlusion, and the resulting damage was observed by staining with 2, 3, 5-triphenylterazolium chloride (TTC). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GM3 and GM1 increased in the damaged cerebral cortex, and immunofluorescence microscopy also revealed a significant change in expression of GM1. In addition, in situ hybridization demonstrated an increase in the mRNA for ganglioside GM3 synthase. These results suggest that gangliosides GM1 and GM3 may be synthesized in vivo to protect the cerebral cortex from ischemic damage.

It has been reported that defects of mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) are involved in many human diseases (such as encephalomyopathies and sporadic Parkinson's disease). However, no effective remedies have been established for complex I deficiencies. We have adopted a gene therapy approach utilizing the NDI1 gene that codes for the single subunit NADH dehydrogenase of Saccharomyces cerevisiae (Ndi1). Our earlier experiments show that the Ndi1 protein can replace or supplement the functionality of complex I in various cultured cells. For this approach to be useful, it is important to demonstrate in vivo that the mature protein is correctly placed in mitochondria. In this study, we have attempted in vivo expression of the NDI1 gene in skeletal muscles and brains (substantia nigra and striatum) of rodents. In all tissues tested, the Ndi1 protein was identified in the injected area by immunohistochemical staining at 1-2 weeks after the injection. Sustained expression was observed for at least 7 months. Double-staining of the sections using antibodies against Ndi1 and F(1)-ATPase revealed that the expressed Ndi1 protein was predominantly localized to mitochondria. In addition, the tissue cells expressing the Ndi1 protein stimulated the NADH dehydrogenase activity, suggesting that the expressed Ndi1 is functionally active. It was also confirmed that the Ndi1 expression induced no inflammatory response in the tissues examined. The data indicate that the NDI1 gene will be a promising therapeutic tool in the treatment of encephalomyopathies and neurodegenerative diseases caused by complex I impairments.

Exposure of rats to the pesticide and complex I inhibitor rotenone reproduces features of Parkinson's disease, including selective nigrostriatal dopaminergic degeneration and alpha-synuclein-positive cytoplasmic inclusions (Betarbet et al., 2000; Sherer et al., 2003). Here, we examined mechanisms of rotenone toxicity using three model systems. In SK-N-MC human neuroblastoma cells, rotenone (10 nm to 1 microm) caused dose-dependent ATP depletion, oxidative damage, and death. To determine the molecular site of action of rotenone, cells were transfected with the rotenone-insensitive single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (NDI1), which incorporates into the mammalian ETC and acts as a "replacement" for endogenous complex I. In response to rotenone, NDI1-transfected cells did not show mitochondrial impairment, oxidative damage, or death, demonstrating that these effects of rotenone were caused by specific interactions at complex I. Although rotenone caused modest ATP depletion, equivalent ATP loss induced by 2-deoxyglucose was without toxicity, arguing that bioenergetic defects were not responsible for cell death. In contrast, reducing oxidative damage with antioxidants, or by NDI1 transfection, blocked cell death. To determine the relevance of rotenone-induced oxidative damage to dopaminergic neuronal death, we used a chronic midbrain slice culture model. In this system, rotenone caused oxidative damage and dopaminergic neuronal loss, effects blocked by alpha-tocopherol. Finally, brains from rotenone-treated animals demonstrated oxidative damage, most notably in midbrain and olfactory bulb, dopaminergic regions affected by Parkinson's disease. These results, using three models of increasing complexity, demonstrate the involvement of oxidative damage in rotenone toxicity and support the evaluation of antioxidant therapies for Parkinson's disease.

The proton-translocating NADH-quinone oxidoreductase in mitochondria (complex I) and bacteria (NDH-1) was shown to be inhibited by amiloride derivatives that are known as specific inhibitors for Na(+)/H(+) exchangers. In bovine submitochondrial particles, the effective concentrations were about the same as those for the Na(+)/H(+) exchangers, whereas in bacterial membranes the inhibitory potencies were lower. These results together with our earlier observation that the amiloride analogues prevent labeling of the ND5 subunit of complex I with a fenpyroximate analogue suggest the involvement of ND5 in H(+) (Na(+)) translocation and no direct involvement of electron carriers in H(+) (Na(+)) translocation.

Numerous studies suggest that dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I) is associated with neurodegenerative disorders, such as Parkinson's disease and Huntington's disease. Development of methods to correct complex I defects seems important. We have previously shown that the single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (Ndi1P) can work as a replacement for complex I in mammalian cells. Using a recombinant adeno-associated virus vector carrying the NDI1 gene, we now demonstrated that the Ndi1 enzyme was successfully expressed in the dopaminergic cell lines rat PC12 and mouse MN9D. The cells expressing the Ndi1 protein were resistant to known inhibitors of complex I, such as rotenone and pyridaben. In addition, the NDI1-transduced cells were still capable of morphological maturation as examined by induction of neurite outgrowth. Also, it was possible to infect the cells after the maturation. The expressed Ndi1 protein was located both in cell bodies and in neurites and was functionally active. It is conceivable that the NDI1 gene will be a promising tool in the treatment of neurodegenerative conditions caused by complex I inhibition.

Two rye genome-specific random amplified polymorphic DNA (RAPD) markers were identified for detection of rye introgression in wheat. Both markers were amplified in all of the tested materials that contained rye chromatin such as rye, hexaploid triticale, wheat-rye addition lines, and wheat varieties with 1BL.1RS translocation. Two cloned markers, designated pSc10C and pSc20H, were 1012 bp and 1494 bp, respectively. Sequence analysis showed that both pSc10C and pSc20H fragments were related to retrotransposons, ubiquitously distributed in plant genomes. Using fluorescence in situ hybridization (FISH), probe pSc10C was shown to hybridize predominantly to the pericentromeric regions of all rye chromosomes, whereas probe pSc20H was dispersed throughout the rye genome except at telomeric regions and nucleolar organizing regions. The FISH patterns showed that the two markers should be useful to select or track all wheat-rye translocation lines derived from the whole arms of rye chromosomes, as well as to characterize the positions of the translocation breakpoints generated in the proximal and distal regions of rye arms.

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

Onion (Allium cepa L.; 1C=15,000 Mb) is an agriculturally important plant. The genome of onion has been extensively studied at the conventional cytogenetic level, but molecular analyses have lagged behind due to its large genome size. To overcome this bottleneck, a partial bacterial artificial chromosome (BAC) library of onion was constructed. The average insert size of the BAC library was about 100 kb. A total of 48,000 clones, corresponding to 0.32 genome equivalent, were obtained. Fluorescent in situ hybridization (FISH) screening resulted in identification of BAC clones localized on centromeric, telomeric, or several limited interstitial chromosomal regions, although most of the clones hybridized with entire chromosomes. The partial BAC library proved to be a useful resource for molecular cytogenetic studies of onion, and should be useful for further mapping and sequencing studies of important genes of this plant. BAC FISH screening is a powerful method for identification of molecular cytogenetic markers in large-genome plants.

A 314-bp tandemly repeated DNA sequence, named pAc074, was characterized in Allium cepa by fluorescence in situ hybridization (FISH) analyses using random amplified fragment as probe. The nucleotide sequences of the clone pAc074 is partially homologous to the satellite DNA sequences, ACSAT1, ACSAT2, and ACSAT3, of A. cepa with 81%, 81% and 78% similarity, respectively. Our sequential C-banding and FISH with pAc074 probe also clearly showed a close relation between Cheterochromatin at telomeric region and pAc074 sequences on all the chromosomes except on chromosome 6. On the long arm of chromosome 7, pAc074 sequences appeared as interstitial band which did not correspond to C-heterochromatin bands. Instead, the C-heterochromatin bands corresponded with the 5S rDNA signals. This is the first evidence of simultaneous banding of the 5S rDNA and C-band in A. cepa.

The Ndi1 enzyme of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. We have shown previously that the NDI1 gene can be functionally expressed in Chinese hamster cells (Seo, B. B., Kitajima-Ihara, T., Chan, E. K., Scheffler, I. E., Matsuno-Yagi, A., and Yagi, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9167-9171) and human embryonal kidney 293 (HEK 293) cells (Seo, B. B., Matsuno-Yagi, A., and Yagi, T. (1999) Biochim. Biochem. Acta 1412, 56-65) and that the Ndi1 protein is capable of compensating respiratory deficiencies caused by defects in the host NADH-quinone oxidoreductase (complex I). To extend the potential use of this enzyme to repair complex I deficiencies in vivo, we constructed a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1). With rAAV-NDI1 as the gene delivery method, we were able to achieve high transduction efficiencies (nearly 100%) even in 143B cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods. The NDI1 gene was successfully introduced into non-proliferating human cells using rAAV-NDI1. The expressed Ndi1 protein was shown to be functionally active just as seen for proliferating cells. Furthermore, when cells were cultured under the conditions where energy has to be provided by respiration, the NDI1-transduced cells were able to grow even in the presence of added complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. In contrast, control cells that did not receive the NDI1 gene failed to survive as anticipated. The Ndi1 protein has a great potential as a molecular remedy for complex I defects, and it is highly likely that the same strategy can be extended to correction of other mitochondrial disorders.

The MWFE polypeptide of mammalian complex I (the proton-translocating NADH-quinone oxidoreductase) is 70 amino acids long, and it is predicted to be a membrane protein. The NDUFA1 gene encoding the MWFE polypeptide is located on the X chromosome. This polypeptide is 1 of approximately 28 "accessory proteins" identified in complex I, which is composed of 42 unlike subunits. It was considered accessory, because it is not one of the 14 polypeptides making up the core complex I; a homologous set of 14 polypeptides can make a fully functional proton-translocating NADH-quinone oxidoreductase in prokaryotes. One MWFE mutant has been identified and isolated from a collection of respiration-deficient Chinese hamster cell mutants. The CCL16-B2 mutant has suffered a deletion that would produce a truncated and abnormal MWFE protein. In these mutant cells, complex I activity is reduced severely (<10%). Complementation with hamster NDUFA1 cDNA restored the rotenone-sensitive complex I activity of these mutant cells to approximately 100% of the parent cell activity. Thus, it is established that the MWFE polypeptide is absolutely essential for an active complex I in mammals.

Chromosomal localizations and distribution patterns of the 5S rRNA genes by means of fluorescence in-situ hybridization in diploid Allium species could help to classify species into chromosome types and aid in determining relationships among genomes. All eleven diploid species were classified into five types, A to E. Species of type A showed a pair of 5S rRNA signals on the short arm of chromosome 5 and two pairs of signals on both arms of chromosome 7. Species of types B and C showed one pair and two pairs of signals on the short arm of chromosome 7, respectively. Type D species showed two pairs of signals on the satellite region of the short arm and a pair of signals on the long arm of chromosome 7. Type E species showed three distinct 5S rRNA gene loci signals on the short arm of chromosome 7. Information on chromosomal localization of 5S rRNA gene loci and distribution patterns within chromosomes in diploid Allium species could help to infer the pathway of origin of the three kinds of alloploid species. Data indicate that A. wakegi is an allopolyploid with genomes of types B and C, and A. deltoide-fistulosum is an allotetraploid derived from a natural hybridization between different species within chromosome type A. Results indicate that A. senescens is an allopolyploid with type B chromosomes and an unidentified chromosomal type.

The NDI1 gene encoding rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria was cotransfected into the complex I-deficient Chinese hamster CCL16-B2 cells. Stable NDI1-transfected cells were obtained by screening with antibiotic G418. The NDI1 gene was shown to be expressed in the transfected cells. The expressed Ndi1 enzyme was recognized to be localized to mitochondria by immunoblotting and confocal immunofluorescence microscopic analyses. Using digitonin-permeabilized cells, it was shown that the transfected cells, but not nontransfected control cells, exhibited the electron transfer activities with glutamate/malate as the respiratory substrate. The activities were inhibited by flavone, antimycin A, and KCN but not by rotenone. Added NADH did not serve as the substrate, suggesting that the expressed Ndi1 enzyme was located on the matrix side of the inner mitochondrial membranes. Furthermore, although nontransfected cells could not survive in a medium low in glucose (0.6 mM), which is a substrate of glycolysis, the NDI1-transfected cells were able to grow in the absence of added glucose. When glycolysis is slow, either at low glucose concentrations or in the presence of galactose, respiration is required for cells to survive. The mutant cells do not survive at low glucose or in galactose, but they can be rescued by Ndi1. These results indicated that the S. cerevisiae Ndi1 was expressed functionally in CCL16-B2 cells and catalyzed electron transfer from NADH in the matrix to ubiquinone-10 in the inner mitochondrial membranes. It is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.