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Parkinson’s disease and other neurological disorders are prevalent in Mongolia. Our previous studies revealed a significant correlation of these diseases with high oxidative stress due to a high body burden of harmful metals, such as manganese, iron, lead, cadmium, and aluminum. This report describes a 37-year-old male Mongolian patient with multiple sclerosis and essential micronutrient deficiency. This patient demonstrated high oxidative stress, as shown by high urinary 8-hydroxy-2'-deoxyguanosine levels of 14.7 and 14.3 ng/mg creatinine (crea), although his hair levels of these toxic metals were markedly lower than other Mongolians. In addition, this patient was deficient not only in various essential minerals, including selenium, magnesium, copper, cobalt, vanadium, and nickel, but also in micronutrients such as vitamin B6, C, E, folic acid, niacin, and β-carotene. Furthermore, after taking 2,3-dimercaptosuccinic acid, a chelating agent, urinary excretion of lead, cadmium, manganese, aluminum, iron, copper, and lithium were increased 156-, 8.4-, 7.6-, 4.3-, 3.3-, 2.1-, and 2.1-fold, respectively. These results suggest that this patient suffered from a deficiency in micronutrients such as essential minerals and vitamins, which resulted in a disturbance in the ability to excrete harmful metals into the urine and hair. It is possible that a deficiency of micronutrients and a high burden of heavy metals play a role in the pathogenesis of multiple sclerosis. Nutritional treatment may be an effective approach to this disease.

The Republic of Palau belongs to Micronesia, and obese people and lifestyle-related diseases are prevalent there. We investigated the relationship of dietary habits and obesity to oxidative stress in Palauan people, as compared with those of Japanese and Mongolian people. A total of 126 healthy Palauan subjects were enrolled. Oxidative stress was evaluated by serum level of reactive oxygen metabolites (ROM). Antioxidant capacity was evaluated by serum level of biological antioxidant potential (BAP). In Palauan subjects, BMI30 was observed in 45.0% of males and 59.1% of females (Japanese: males 1.3%, females 0.8%, Mongolian: males 6.3%, females 14.7%). Palauan subjects consumed 2553 kcal per day (Japanese 2121 kcal, Mongolians 2534 kcal). The ratios of carbohydrate energy to total energy were 59.8 % (Japanese 54.7 %, Mongolians 50.2%). The ratios of fat energy to total energy were 22.9% (Japanese 26.7%, Mongolians 34.5%). ROM levels in Palauan subjects showed higher than those in Japanese subjects, while BAP levels of Palauan subjects did not decrease compared to those of Japanese. ROM levels correlated with body fat ratio, and showed a reverse correlation with handgrip strength. Handgrip strength decreased in the subjects of more than forty years of age. These findings suggest that the obesity in Palauan people may have a connection with high intake of calories through carbohydrate eating rather than through fat eating. Their high oxidative stress may be induced by obesity, and contribute to an early decline of handgrip strength, ultimately in early aging.

Mongolians are known to have relatively short life expectancy. In order to examine the role of dietary habits in the early aging of Mongolians, the food intake inquiry, anthropometric measurements and blood clinical tests were performed for 365 healthy inhabitants in Murun, a northern Mongolia city, and compared to those of Japanese. Murun inhibitants were found to have a characteristic dietary habit of taking large amounts of meat, milk, dairy products and wheat flour products, in contrast little vegetables, fruits and fishes. The daily calorie intake of the adults was estimated to be 2,525 kcal, and the fat/total calorie ratio was calculated 33.7%, about 1.3-fold higher than that of Japanese. The intake ratio of fatty acid from the Mongolian foods, saturated : mono-unsaturated : poly-unsaturated fatty acids (PUFA) ratio, was 10.3 : 7.8 : 3.0. Results of blood clinical tests showed significantly higher levels of serum triglycerides, low-density lipoprotein cholesterol (LDL) and homocysteine, and lower levels of high-density lipoprotein cholesterol (HDL), n-3 PUFA, folic acid and adiponectin, in comparison with those of Japanese. In addition, the Mongolians were also found to have significantly high levels of oxidative stress markers, such as serum malondialdehyde-modified LDL (MDA-LDL), urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and serum reactive oxygen metabolites (ROM). The serum ROM level in the Mongolians seemed to associate with their body fat ratio (p < 0.05), and was significantly inverse-correlated to handgrip strength (p < 0.001). Obesity was observed at a high incidence in the subjects over 30-year old, and over 40-year old their handgrip ability was markedly decreased. These findings suggest that in the Mongolians the dietary habits associate with their lifestyle-related diseases and early aging, and the improvement of dietary habits is an effective strategy for health promotion of the inhabitants.

A previous study revealed a high prevalence of hepatitis B surface antigen (HBsAg) and hepatitis delta virus (HDV) RNA among 249 apparently healthy individuals (mean ± standard deviation age, 48.4 ± 13.9 years; 126 males and 123 females) in Ulaanbaatar, Mongolia. To investigate further the prevalence of HDV infection there, the same serum samples obtained from the cohort were tested for the presence of immunoglobulin G (IgG) class antibody to HDV (anti‐HDV) by a newly developed enzyme‐linked immunosorbent assay using recombinant hepatitis delta antigen protein expressed in the pupae of silkworm as the antigen probe. Anti‐HDV was detected in 42 persons (16.9%), among whom 22 (52.4%) were positive for HBsAg and 20 (47.6%) had detectable HDV RNA. Among 170 persons with anti‐HBc in the absence of HBsAg, 20 (11.8%) tested positive for anti‐HDV, and 1 of the 20 subjects was positive for HDV RNA. Of note, none of 55 anti‐HBc‐negative persons had anti‐HDV, supporting the specificity of the anti‐HDV assay. The optical density (OD) value of anti‐HDV was significantly higher among HDV RNA‐positive subjects (n = 21) than among HDV RNA‐negative subjects (n = 21) (2.513 ± 0.514 vs. 0.836 ± 0.550, P< 0.0001). The present study confirmed the extremely high prevalence of HDV infection in Mongolia, and identified a person who was positive for both anti‐HDV and HDV RNA despite negativity for HBsAg and HBV DNA probably due to viral interference. The anti‐HDV assay may be useful for further epidemiological studies on HDV infection in larger cohorts in urban and rural areas of Mongolia, where elucidation of the transmission route of HDV is required urgently. J. Med. Virol. 76:333–340, 2005. © 2005 Wiley‐Liss, Inc.

ABSTRACTThe prevalence of infection with hepatitis A virus (HAV), HBV, HCV, HDV, and HEV was evaluated in 249 apparently healthy individuals, including 122 inhabitants in Ulaanbaatar, the capital city of Mongolia, and 127 age- and sex-matched members of nomadic tribes who lived around the capital city. Overall, hepatitis B surface antigen (HBsAg) was detected in 24 subjects (10%), of whom 22 (92%) had detectable HBV DNA. Surprisingly, HDV RNA was detectable in 20 (83%) of the 24 HBsAg-positive subjects. HCV-associated antibodies were detected in 41 (16%) and HCV RNA was detected in 36 (14%) subjects, none of whom was coinfected with HBV, indicating that HBV/HCV carriers account for one-fourth of this population. Antibodies to HAV and HEV were detected in 249 (100%) and 28 (11%) subjects, respectively. Of 22 HBV DNA-positive subjects, genotype D was detected in 21 subjects and genotype F was detected in 1 subject. All 20 HDV isolates recovered from HDV RNA-positive subjects segregated into genotype I, but these differed by 2.1 to 11.4% from each other in the 522- to 526-nucleotide sequence. Of 36 HCV RNA-positive samples, 35 (97%) were genotype 1b and 1 was genotype 2a. Reflecting an extremely high prevalence of hepatitis virus infections, there were no appreciable differences in the prevalence of hepatitis virus markers between the two studied populations with distinct living place and lifestyle. A nationwide epidemiological survey of hepatitis viruses should be conducted in an effort to prevent de novo infection with hepatitis viruses in Mongolia.

Abstract: The photoaffinity spin-labeled ATP analog, 2-N3-SL-adenosine triphosphate (ATP), was used to covalently modify isolated β-subunits from F1-ATPase of the thermophilic bacterium PS3. Approximately 1.2 mol of the nucleotide analog bound to the isolated subunit in the dark. Irradiation leads to covalent incorporation of the nucleotide into the binding site. ESR spectra of the complex show a signal that is typical for protein-immobilized radicals. Addition of isolated α-subunits to the modified β-subunits results in ESR spectra with two new signals indicative of two distinctly different environments of the spin-label, e.g., two distinctly different conformations of the catalytic sites. The relative ratio of the signals is approx 2∶1 in favor of the more closed conformation. The data show for the first time that when nucleotides are bound to isolated β-subunits, binding of α-subunits induces asymmetry in the catalytic sites even in the absence of the γ-subunit.

Mitochondrial DNA (mtDNA) is highly susceptible to mutations that result in polymorphisms and diseases including diabetes. We analyzed heteroplasmy, polymorphisms related to diabetes, and complementation by fusogenic proteins. Cytoplast fusion and microinjection allow, defects in mutated mtDNA inside a heteroplasmic cell to be complemented by fusing two mitochondria via human fusogenic proteins. We characterized three hfzos as well as two OPA1s that prevent apoptosis. Two coiled coil domains and GTPase domains in these fusogenic proteins regulate membrane fusion. The hfzo genes were expressed mainly in the brain and in muscle that are postmitotic, but not in the pancreas. Under the in.uence of polymorphisms of mtDNA and nDNA, the vicious circle of reactive oxygen species and mutations in cell can be alleviated by mitochondrial fusion.

Mitochondrial DNA (mtDNA) is highly susceptible to mutations that result in polymorphisms and diseases including diabetes. We analyzed heteroplasmy, polymorphisms related to diabetes, and complementation by fusogenic proteins. Cytoplast fusion and microinjection allow, defects in mutated mtDNA inside a heteroplasmic cell to be complemented by fusing two mitochondria via human fusogenic proteins. We characterized three hfzos as well as two OPA1s that prevent apoptosis. Two coiled coil domains and GTPase domains in these fusogenic proteins regulate membrane fusion. The hfzo genes were expressed mainly in the brain and in muscle that are postmitotic, but not in the pancreas. Under the in.uence of polymorphisms of mtDNA and nDNA, the vicious circle of reactive oxygen species and mutations in cell can be alleviated by mitochondrial fusion.

Mitochondrial ATP synthase γ-subunit (F1γ) pre- mRNA undergoes alternative splicing in a tissue- or cell type-specific manner. Exon 9 of F1γ pre-mRNA is specifically excluded in heart and skeletal muscle tissues and in acid-stimulated human fibrosarcoma HT1080 cells, rhabdomyosarcoma KYM-1 cells, and mouse myoblast C2C12 cells. Recently, we found a purine-rich exonic splicing enhancer (ESE) element on exon 9 via transgenic mice bearing F1γ mutant minigenes and demonstrated that this ESE functions ubiquitously with exception of muscle tissue (Ichida, M., Hakamata, Y., Hayakawa, M., Ueno E., Ikeda, U., Shimada, K., Hamamoto, T., Kagawa, Y., Endo, H. (2000) J. Biol. Chem.275, 15992–16001). Here, we identified an exonic negative regulatory element responsible for muscle-specific exclusion of exon 9 using bothin vitroand in vivosplicing systems. A supplementation assay with nuclear extracts from HeLa cells and acid-stimulated HT1080 cells was performed for an in vitroreaction of muscle-specific alternative splicing of F1γ minigene and revealed that the splicing reaction between exons 8 and 9 was the key step for regulation of muscle-specific exon exclusion. Polypyrimidine tract in intron 8 requires ESE on exon 9 for constitutive splice site selection. Mutation analyses on the F1γEx8-9 minigene using a supplementation assay demonstrated that the muscle-specific negative regulatory element is positioned in the middle region of exon 9, immediately downstream from ESE. Detailed mutation analyses identified seven nucleotides (5′-AGUUCCA-3′) as a negative regulatory element responsible for muscle-specific exon exclusion. This element was shown to cause exon skipping in in vivosplicing systems using acid-stimulated HT1080 cells after transient transfection of several mutant F1γEx8-9-10 minigenes. These results demonstrated that the 5′-AGUUCCA-3′ immediately downstream from ESE is a muscle-specific exonic splicing silencer (MS-ESS) responsible for exclusion of exon 9in vivoand in vitro.

A novel photoaffinity label, 8- N 3 -3′-biotinyl-ATP, has been synthesized. The introduction of an additional biotin residue is advantageous for easy detection of labeled proteins. This could be first tested by reaction with the F 1 -ATPase from the thermophilic bacterium PS3 (TF 1 ). UV irradiation of TF 1 in the presence of 8- N 3 -3′-biotinyl-ATP results in a nucleotide-dependent binding of the analogue in the noncatalytic α and the catalytic β subunits of TF 1 , demonstrating the suitability of this analogue as a potential photoaffinity label. Reaction with the V 1 -ATPase, however, led to labeling of subunit E, which has been suggested as a structural and functional homologue of the γ subunit of the F-ATPases. MALDI-TOF mass spectrometry has been used to map the regions of subunit E involved in the binding of 8- N 3 -3′-biotinyl-ATP.

A novel photoaffinity label, 8-N₃-3'-biotinyl-ATP, has been synthesized. The introduction of an additional biotin residue is advantageous for easy detection of labeled proteins. This could be first tested by reaction with the F₁-ATPase from the thermophilic bacterium PS3 (TF₁). UV irradiation of TF₁ in the presence of 8-N₃-3'-biotinyl-ATP results in a nucleotide-dependent binding of the analogue in the noncatalytic α and the catalytic β subunits of TF₁, demonstrating the suitability of this analogue as a potential photoaffinity label. Reaction with the V₁-ATPase, however, led to labeling of subunit E, which has been suggested as a structural and functional homologue of the γ subunit of the F-ATPases. MALDI-TOF mass spectrometry has been used to map the regions of subunit E involved in the binding of 8-N₃-3'-biotinyl-ATP. Copyright 2001 Academic Press.

We examined the genetic contribution of single nucleotide polymorphisms (SNPs) of the energy metabolism-related genes, including beta 3 adrenergic receptor (β3AR), apolipoprotein E (apo-E), promoter of uncoupling protein 3 (UCP3-p), peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and leptin receptor (LEPR) to metabolic disorders, in 118 inhabitants of Palau. The data were statistically analyzed and ethnically compared to correlate SNPs and their metabolic parameters. UCP3-p (P < 0.01) and PPARγ2 (p = 0.05) correlated with plasma HbA1c, and UCP3-p correlated with fasting blood glucose (P < 0.01) in males, but not in females. UCP3-p correlated with body fat (%) (P < 0.01) in females, but not in males. Plasma leptin levels and apo-E were correlated in both groups. The frequency of SNPs for PPARγ2, LEPR, and UCP3-p are significantly different between Palauans and Caucasians.

Colocalization of mitochondria is the first step of intermitochondrial interaction or fusion in a cell. Here, we showed colocalization between exogenous mitochondria and endogenous ones or between exogenous proteoliposomes and endogenous mitochondria in mouse fertilized eggs by confocal laser microscopy. Isolated mitochondria from mouse liver and proteoliposomes containing mitochondrial membrane were directly labeled with red fluorescent aliphatic marker, PKH26, which is incorporated into lipid membrane, and then were microinjected into fertilized mouse eggs. Exogenous mitochondria appeared to be almost colocalized with endogenous mitochondria at the 4- and 8-cell stages, when mitochondria were stained with Rhodamine 123 (green fluorescent marker). On the contrary, when liposomes consisted of soy bean phospholipid were microinjected into the eggs as a control, their localization was different from that of endogenous mitochondria. Next, the submitochondrial particles and proteoliposomes were microinjected. Both the proteoliposomes and the submitochondrial particles appeared to colocalize with endogenous mitochondria at the 4-cell stage. These results suggest the existence of a factor that makes liposomes colocalize with mitochondria. Such a proteoliposome would be useful for the development of mitochondrial gene transfer techniques.

Ganglioside GD3 induced the release of cytochrome c from isolated rat liver mitochondria. This process was completely prevented by cyclosporin A and partially prevented by a cysteine protease inhibitor, n-acetyl-leu-leu-norleucinal. Cyclosporin A is a potent inhibitor of the permeability transition pore, whereas n-acetyl-leu-leu-norleucinal has no effect on this pore. These results indicate that the release of cytochrome c from mitochondria requires both the opening of the permeability transition pore and a cysteine protease inhibitor-sensitive mechanism. Gangliosides GD1a, GD1b, GT1b, and GQ1b along with the synthetic GD3 mimetics TMS-42 and CI-22, which are glycerophospholipids carrying a disialo residue, also induced cytochrome c release. In contrast, gangliosides GM1, GM2, and GM3 did not induce cytochrome c release. These results indicate that two sialo residues must play an important role in the induction of cytochrome c release by gangliosides.

ATP synthase (FoF1) consists of F1(ATP-driven motor) and Fo(H+-driven motor). F1is a complex of α3β3γδε subunits, and γ is the rotating cam in α3β3. Thermophilic F1(TF1) is exceptional in that it can be crystallized as a β monomer and an α3β3oligomer, and it is sufficiently stable to allow αβ refolding and reassembly of hybrid complexes containing 1, 2, and 3 modified α or β. The nucleotide-dependent open–close conversion of conformation is an inherent property of an isolated β and energy and signals are transferred through α/β interfaces. The catalytic and noncatalytic interfaces of both mitochondrial F1(MF1) and TF1were analyzed by an atom search within the limits of 0.40 nm across the αβ interfaces. Seven (plus thermophilic loop in TF1) contact areas are located at both the catalytic and noncatalytic interfaces on the open β form. The number of contact areas on closed β increased to 11 and 9, respectively, in the catalytic and noncatalytic interfaces. The interfaces in the barrel domain are immobile. The torsional elastic strain applied through the mobile areas is concentrated in hinge residues and the P-loop in β. The notion of elastic energy in FoF1has been revised. X-ray crystallography of F1is a static snap shot of one state and the elastic hypotheses are still inconsistent with the structure, dyamics, and kinetics of FoF1. The domain motion and elastic energy in FoF1will be elucidated by time-resolved crystallography.

Muscle-specific isoform of the mitochondrial ATP synthase γ subunit (F1γ) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novoprotein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem.273, 8492–8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998)Biochem. Biophys. Res. Commun.251, 603–608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F1γ gene by an in vivosplicing system using cultured cells and transgenic mice. We constructed a F1γ wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F1γ Pu-del and F1γ Pu-rich mutants, respectively. Based on an in vivosplicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F1γ Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F1γ Pu-rich minigene mice revealed that the F1γ Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F1γ pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.

Recent advances in bioenergetics consist of discoveries related to rotational coupling in ATP synthase (FoF1), uncoupling proteins (UCP), reactive oxygen species (ROS) and mitochondrial DNA (mtDNA). As shown in cloned sheep, mammalian genomes are composed of both nuclear DNA (nDNA) and maternal mtDNA. Oxidative phosphorylation (oxphos) varies greatly depending on cellular activities, and is regulated by both gene expression and the electrochemical potential difference of H+ (Δ μH+). The expression of both mtDNA (by mtTFA) and nDNA for oxphos and UCP (by NRFs, etc.) is coordinated by a factor called PGC-1. The Δ μH+ rotates an axis in FoF1 that is regulated by inhibitors and ATP-sensitive K+-channels. We cultured human ρo cells (cells without mtDNA) in synthetic media and elucidated relationships among mtDNA, nDNA, Δ μH+, UCPs, ROS, and apoptosis. These cells lack oxphos-dependent ROS formation and survive under conditions of high O2. Cells cultured in the absence of ROS scavengers have proliferated for 40 years. UCPs lower Δ μH+ and prevent ROS formation and resulting apoptosis. These results were applied to diabetology and gerontology. The pancreatic ρo cells did not secrete insulin, and mtDNA mutations caused diabetes, owing to the deficient Δ μH+. Insulin resistance was closely related to UCPs and other energy regulators. The resulting high-glucose environment caused glycation of proteins and ROS-mediated apoptosis in vascular cells involved in diabetic complications. Telomeres, oxphos, and ROS are determinants in cellular aging. Cell division and ROS shortened telomeres and accelerated aging. In aged cells, Δ μH+ was reduced by the slow respiration, and this change induced apoptosis. Cybrids made from aged cytoplasts and ρo cells showed that both decreased expression of nDNA, and somatic mutations of mtDNA are involved in the slowing of respiration in aged cells.

The effects of n-3 polyunsaturated fatty acids (n-3PUFA) on obesity and diabetes were examined using KK-Aymice fed with perilla oil (P), soybean oil (S), or lard (L), and those containing 30% fish oil (PF, SF, or LF), containing eicosapentaenoic acid (EPA = 9.9%) and docosahexaenoic acid (DHA = 18.0%). Perilla oil contained the largest proportion of linolenic acid (LNA = 61.9%). Computerized tomography (CT) scans showed narrower areas of visceral fat in the abdominal cross sections of groups given fish oil (PF, SF, and LF) and lower leptin levels (p < 0.05-p < 0.001) compared with controls (P, S, and L), without significant changes in energy intake and body weight. The highest plasma n-3PUFA content (21.31 ± 0.35%) was attained with PF. This group contained 2.6-fold more plasma DHA (p < 0.001), and expressed 2.7-fold more UCP2 mRNA in white adipose tissue (p < 0.01) than in the P group. The epididymal fat pad (p < 0.05) weighed less, and levels of blood glucose (p < 0.05) and total cholesterol (p < 0.01) were reduced in PF compared with P.

Culture media for mammalian cells contain electrolytes at concentrations that approximate those of the extracellular fluid (ECF). However, we observed that HeLa strain cells can grow in a cytosol type, serum-free, high amino acid medium (KN13) in which the concentrations of K+, Mg2+and Pi were increased to about 10 times those in ECF by replacing NaCl. The Ca2+concentration of KN13 is 10% of that in ECF. The additions of Mg2+and Pi are essential to prevent the harmful effect of a high concentration of K+in the medium. Even an increase in the KNaratio to 7 and 9 permitted growth for at least 2 weeks and 4 days, respectively. HeLaP3 cells adapted to KN13 (HeLaK cells) have proliferated (4 fold/week) for 4 years in this medium. The Na,K-ATPase of HeLaK cells was the α1 isoform. HeLaK cells in KN13 medium have a lower membrane potential (−3.0 ± 6.1 mV) than that of the original HeLaP3 cells in DM160 (−26 ± 10 mV).

To localize the nucleotide binding sites of the F1ATPase (TF1) from the thermophilic bacterium PS3 we have used 14C‐labeled 8‐azido ATP (8‐N3ATP) as photoaffmity label. 8‐N3ATP is hydrolyzed by the F,ATPase in the absence of ultraviolet light. Irradiation by ultraviolet light of the enzyme in the presence of 8‐N3ATP results in reduction of ATPase activity and in preferential nucleotide specific labeling of the α subunits (0.8–0.9 mol 8‐N3ATP/TF1,α:β = 4:1). Inactivation and labeling do not depend on the presence of Mg2+. Both effects decrease upon addition of various nucleotide di‐ or triphosphates.

Summary Measurements were made of the difference in the electrochemical potential of protons ( ) across the membrane of vesicles reconstituted from the ATPase complex (TF0·F1) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential (??) and pH difference across the membrane (? pH), respectively.

Summary Biomembranes are the major site of energy transduction. The chemisomotic theory of energy transduction is based on the following four major systems (i) H+-ATPase which is composed of a catalytic portion (F1) and a H+-channel (Fo), (ii) electron transport components, (iii) H+-linked porters, and (iv) a H+-impermeable lipid bilayer which is plugged through by systemsi toiii that are specially oriented to translocate H+.

Tissue‐specific isoforms of the γ‐subunit of the bovine F0F1‐ATP synthase were identified for the first time. The two isoforms, heart and liver type, were generated by alternative splicing, the liver‐type RNA transcript containing a 37‐nucleotide sequence as a cassette exon. Protein sequencing of the C‐terminal fragments of the two isoforms of the F1γ‐subunit indicated that the liver‐type isoform had an additional aspartate residue at the C terminus, not present in the heart type one.

ATP synthase (F0F1) is driven by an electrochemical potential of H+ (δΜH+). F0F1 is composed of an ion-conducting portion (F0) and a catalytic portion (F1). The subunit composition of F1 is α3β3γδε. The active α3β3 oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F1 (TF1). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the βDELSEED sequence via γδε-F0. In fact, cross-linking of βDELSEED to γ stopped the ATP-driven rotation of γ in the center of α3β3. The torque of the rotation is estimated to be 420 pN·å from the δΜH+ and H+-current through F0F1. The angular velocity (Ω) of γ is the rate-limiting step, because δΜH+ increased theVmax of H+ current through F0, but not theKm(ATP). The rotational unit of F0 (=ab2c10) is π/5, while that in α3β3 is 2π/3. This difference is overcome by an analog-digital conversion via elasticity around βDELSEED with a threshold to release ATP. The αβ distance at the C site is about 9.6 å (2,8-diN3-ATP), and tight Mg-ATP binding in α3β3γ was shown by ESR. The rotational relaxation of TF1 is too rapid (Φ=100 nsec), but the rate of AT(D)P-induced conformational change of α3β3 measured with a synchrotron is close to Ω. The ATP bound between the P-loop and βE188 is released by the shift of βDELSEED from γRGL. Considering the viscosity resistance and inertia of the free rotor (γ-c), there may be a stator containing OSCP (=δ of TF1) and F0-d to hold free rotation of α3β3.

The ATPase activity of the F1-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 µM where 70% stimulation is observed at 36°C. Half maximal stimulation is observed at about 3 µM dye. At rhodamine 6G concentrations greater than 10 µM, ATPase activity declines with 50% inhibition observed at about 75 µM dye. The ATPase activities of the α3β3γ and α3β3γδ complexes assembled from isolated subunits of TF1 expressed inE. coli deleted of theunc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF1. In contrast, the ATPase activities of the α3β3 and α3β3δ complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 µM dye at 36°C. The ATPase activity of TF1 is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF1 is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 µM dye at 30°C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF1 stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.

A new type of acetylcholine sensor was made with an Ion Sensitive Field Effect Transistor (ISFET) and acetylcholine receptor. The acetylcholine receptor was fixed on a polyvinylbutyral membrane which covered the ISFET gate. When acetylcholine was injected into this system, the differential gate output voltage gradually Shifted to the positive side and reached a constant value. This response was due to the positive charge of acetylcholine. A linear relationship was obtained between the initial rate of the differential gate output voltage change and the logarithmic value of the acetylcholine concentration. Acetylcholine was fixed in the range 0.1-10M. When the acetylcholine receptor was immobilized with the lipid membrane, the response was amplified with both the positive charge of acetylcholine and sodium ion flux through the acetylcholine receptor's channel. Therefore, the difference in the differential voltage between the acetylcholine receptor-ISFET systems with and without the lipid membrane was caused by sodium ion flux through the acetylcholine receptor's channel.

To localize the nucleotide binding sites of the F 1ATPase (TF 1) from the thermophilic bacterium PS3 we have used 14C-labeled 8-azido ATP (8-N 3ATP) as photoaffmity label. 8-N 3ATP is hydrolyzed by the F,ATPase in the absence of ultraviolet light. Irradiation by ultraviolet light of the enzyme in the presence of 8-N 3ATP results in reduction of ATPase activity and in preferential nucleotide specific labeling of the α subunits (0.8–0.9 mol 8-N 3ATP/TF 1,α:β = 4:1). Inactivation and labeling do not depend on the presence of Mg 2+. Both effects decrease upon addition of various nucleotide di- or triphosphates.

The reaction of ATP synthase (F0F1) is the final step in oxidative phosphorylation (OXPHOS). Although OXPHOS has been studied extensively in bacteria, no tissue-specific functions nor bioenergetic disease, such as mitochondrial encephalomyopathy and aging occur in these organisms. Recent developments of the Human Genome Project will become an important factor in the study of mammalian bioenergetics. To elucidate the physiological roles of human F0F1, genes encoding the subunits of F0F1 were sequenced, and their expression in human cells was analyzed. The following results were obtained: A. The roles of the residues in F0F1 are not only to transform the energy of the electrochemical potential (ΔμH+) across the membrane, but also to respond rapidly to the changes in the energy demand by regulating the intramolecular rotation of F0F1 with the ΔμH+ and the inhibitors of the ATPase. B. The roles of the control regions of the F0F1 genes, are to coordinate both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) depending on the energy demand of the cells, especially in muscle. C. The cause of the age-dependent decline of ATP synthesis has been attributed to the accumulation of mutations in mtDNA. However, the involvement of nDNA in the decline is also important because of telomere shortening in somatic cells, and age-dependent mtDNA expression analyzed with ρ° cells (cells without mtDNA).

A single molecule of ATP synthase (F0F1) is by itself a rotary motor, the smallest ever found, and this biomotor is driven by an electrochemical potential of H+(ΔμH+). F0F1 is composed of an ion- conducting portion (F0) and a catalytic portion (F1). The major breakthroughs in studies on the mechanochemical coupling have been the direct observation of the rotation of a stable α3β3γ complex of thermophilic F1 (TF1), and X-ray crystallography of the α3β3γ portion of mitochondrial F1 (MF1) and the α3β3 oligomer of TF1. This review focuses on the dynamics of TF1, demonstrated by a crucial experiment. The torque of the rotation was estimated to be 42 pN·nm from the ΔμH+and frictional force. Important unsolved problems are the crystallography of F0, elastic energy conversion, and the stator and rotor of this biomotor.

To elucidate the function of group II chaperonin, the gene for the chaperonin from the hyperthermophilic archaeum Thermococcusstrain KS-1 was cloned and sequenced. Two distinct genes coding for chaperonin subunits, designated α and β, were obtained, and their deduced amino acid sequences are highly homologous to those of group II chaperonins from other sources. The α and β subunits were individually expressed in Escherichia coli. Both of the recombinant subunits assemble to constitute the homo-oligomeric double-ring complexes, which are prone to form large aggregates. The α aggregate is dissociated into the typical chaperonin ring complex by incubation in buffer containing 15% (v/v) methanol, while the β aggregate cannot be dissociated. At high temperature, both of the recombinant complexes have weak ATPase activities. They are able to arrest refolding of a chemically denatured thermophilic enzyme in the absence of ATP, and refolding is resumed when ATP is supplemented. These results suggest that homo-oligomeric complexes of the archaeal chaperonin have activity.

Tissue-specific alternative RNA splicing in human F1γ pre-mRNA produces muscle- and nonmuscle-type isoforms. Muscle-specific exclusion of exon 9 of the F1γ gene is cell-specifically induced by acidic treatment of human fibrosarcoma HT1080 and rhabdomyosarcoma KYM-1 cells. We constructed an F1γ minigene containing parts of exon 8, intron 8, and exon 9 of the human F1γ gene and then analyzed a negative factor that inhibited inclusion of exon 9 via anin vitrosplicing assay using acid-stimulated HT1080 cell nuclear extract.In vitrosplicing of the F1γ minigene, similarly to the β-globin minigene used as a control, was observed in HeLa cell nuclear extract. Next, we performed supplemental experiments using HeLa and HT1080 cell nuclear extracts. The splicing reaction of the F1γ minigene was specifically inhibited by supplementation with nuclear extract from acid-stimulated HT1080 cells, whereas that of human β-globin was not inhibited. These results indicated that acidic stimulation induced a negative factor that blocked inclusion of alternatively spliced exon in the F1γ minigenein vitro,and a regulatory factor acted in a sequence-specific manner for muscle-specific alternative splicing in F1γ pre-mRNA.

The reaction of ATP synthase (F0F1) is the final step in oxidative phosphorylation (OXPHOS). Although OXPHOS has been studied extensively in bacteria, no tissue-specific functions nor bioenergetic disease, such as mitochondrial encephalomyopathy and aging occur in these organisms. Recent developments of the Human Genome Project will become an important factor in the study of mammalian bioenergetics. To elucidate the physiological roles of human F0F1, genes encoding the subunits of F0F1were sequenced, and their expression in human cells was analyzed. The following results were obtained: A. The roles of the residues in F0F1are not only to transform the energy of the electrochemical potential (ΔμH+) across the membrane, but also to respond rapidly to the changes in the energy demand by regulating the intramolecular rotation of F0F1with the ΔμH+and the inhibitors of the ATPase. B. The roles of the control regions of the F0F1genes, are to coordinate both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) depending on the energy demand of the cells, especially in muscle. C. The cause of the age-dependent decline of ATP synthesis has been attributed to the accumulation of mutations in mtDNA. However, the involvement of nDNA in the decline is also important because of telomere shortening in somatic cells, and age-dependent mtDNA expression analyzed with ρ° cells (cells without mtDNA).

Phosphoinositide-specific phospholipase C (PLC) is a crucial enzyme in transmembrane signaling. A cDNA encoding the putative phospholipase C was cloned from human lymphocytes that were transformed by infection with Epstein-Barr virus. The deduced amino acid sequence of the cDNA accounted for 146.1 kDa of the molecular mass of the complete enzyme and showed 50.2% sequence similarity to bovine brain PLC. This cDNA contained regions, the sequences of which were similar to those of some tyrosine kinase-related oncogene products. Northern blotting demonstrated that the mRNA for this PLC is expressed in human promyelocytic leukemia cells (HL-60). Since the other cloned cDNAs for PLCs could not hybridize with the RNA from this cell, it is strongly suggested that the gene obtained here encodes an additional isozyme of PLC in blood cells.

The photoactivatable bifunctional 3′-arylazido-β-alanyl-2-azido ATP (2,3′-DiN 3ATP) has been applied to study the localization of the nucleotide-binding sites of coupling factor 1 (F 1ATPase, TF 1) from the thermophilic bacterium PS3 by photoaffinity cross-linking. UV irradiation of TF 1in the presence of 2,3′-DiN 3ATP results in the nucleotide-dependent formation of various higher molecular mass cross-links formed by two, three or even four α- and/or β-subunits. The differences observed upon photoaffinity cross-linking by the bifunctional 2-azido ATP or 8-azido ATP analog are discussed. They are probably due to the varied maximal distance between both azido groups, or to the different conformations ( anti/syn) of these analogs. The results confirm our suggestion that several (possibly all) nucleotide-binding sites of F 1ATPases are located at the interfaces between α- and β-subunits.

To demonstrate the direct interfacial position of nucleotide binding sites between subunits of proteins we have synthesized the bifunctional photoaffinity label 2,8-diazidoadenosine 5′-triphosphate (2,8-DiN 3ATP). UV irradiation of the F 1-ATPase (TF 1) from the thermophilic bacterium PS3 in the presence of 2,8-DiN 3ATP results in a nucleotide-dependent inactivation of the enzyme and in a nucleotide-dependent formation of α-βcrosslinks. The results confirm an interfacial localization of all the nucleotide binding sites on TF 1.

A mutant of Lactococcus lactissubsp. lactisC2 with reduced membrane-bound ATPase activity was characterized to clarify its acid sensitivity. The cytoplasmic pH of the mutant was measured in reference to the parental strain under various pH conditions. At low pH, the mutant could not maintain its cytoplasmic pH near neutral, and lost its viability faster than the parental strain. The ATPase activities of cells cultured under neutral and acidic conditions using pH-controlled jar fermentors were measured. The relative ATPase activity of the mutant at pH 7.0 was 42% of the parental strain. At pH 4.5, the parental strain showed an ATPase activity 2.8-fold higher than that at pH 7.0, while the level of increase in the mutant was only 1.6. Northern and Western blot analyses found that at pH 7.0 the transcriptional level and the amount of F1β subunit were similar in both strains, suggesting that the mutant has a defective ATPase structural gene. On the other hand, at pH 4.5 the transcriptional level and the amount of F1β subunit were found to be significantly higher in both strains than those at pH 7.0. From these results, it was suggested that the mutant has a normal regulation system for ATPase gene expression. It was concluded that the mutant is acid sensitive due to its inability to extrude protons out of the cell with defective ATPase under acidic conditions.

(1) Extensive studies on proton-translocating ATPase (H+-ATPase) revealed that H+-ATPase is an energy transforming device universally distributed in membranes of almost all kinds of cells. (2) Crystallization of the catalytic portion (F1) of H+-ATPase showed that F1 is a hexagonal molecule with a central hole. The diameter of F1 is about 90 Å and its molecular weight is about 380,000. (3) Use of thermophilic F1 permits the complete reconstitution of F1 from its five subunits (a, ß, ?, d, and e) and demonstration of the gate function of the ?de-complex, the catalytic function of ß (supported by a and ?), and the H+-translocating functions of all five subunits. (4) Studies using purified thermostable F0 showed that F0 is an H+-channel portion of H+-ATPase. The direct measurement of H+-flux through F0, sequencing of DCCD-binding protein, and isolation of F1-binding protein are described. (5) The subunit stoichiometry of F1 may be a3ß3?de. (6) Reconstitution of stable H+-ATPase-liposomes revealed that ATP is directly synthesized by the flow of H+ driven by an electrochemical potential gradient and that H+ is translocated by ATP hydrolysis. This rules out functions for all the hypothetical components that do not belong to H+-ATPase in H+-driven ATP synthesis. The roles of conformation change and other phenomena in ATP synthesis are also discussed.

The basic structures of the catalytic portion (F1, a3ß3?de) of ATP synthase are the a3ß3 hexamer (oligomer with cooperativity) and a1ß1 heterodimer (protomer). These were reconstituted from the a and ß subunits of thermophilic F1 (TF1), and the a3ß3 hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F1 and TF1, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.

As a first step in the isolation of a stable Na+/H+ antiporter, its reaction in sonicated membrane vesicles of thermophilic bacterium PS3 has been characterized. The sonicated vesicles showed quenching of quinacrine fluorescence in either NADH oxidation or ATP hydrolysis. The quenching was reversed by the addition of Na+, Li+, Mn2+, Cd2+, and Co2+, but not of choline+ or Ca2+, regardless of their counter anions.22Na+ was taken up into the vesicles by NADH oxidation, and the22Na+ uptake was inhibited by the addition of an uncoupler. H+ release was observed on addition of Na+ to sonicated vesicles. The magnitude of the pH difference across the membrane induced by NADH oxidation was constant at pH 7.0 to 9.1, but the Na+/H+ antiport was affected by the pH of the medium (optimum pH=8.5). TheKm's of the antiporter for Na+ and Li+ were 2.5 and 0.1 mM, respectively, but theVmax values for the two ions were the same at pH 8.0. In the presence of Li+, no further decrease of fluorescence quenching was observed on addition of Na+ andvice versa. The Na+/H+ antiporter activity in PS3 was stable at 70°C, and the optimum temperature for activity was 55–60°C. In contrast to mesophilic cation/H+ antiporters, this antiporter was not inhibited by a thiol reagent.

Mitochondrial hinge protein is a subunit of ubiquinol-cytochrome- creductase in the respiratory chain and ‘hinges’ cytochrome cwith cytochrome c1. The protein is encoded in the nuclear genome, synthesized in the cytosol and then imported into the mitochondria. The cDNA of the human hinge protein has been cloned and its nucleotide sequence was determined. The deduced primary structure of the amino-terminal presequence consists of 13 amino acid residues, of which 4 amino acids are acidic and only one is basic. Since the presequences of most other precursors are rich in basic amino acids, this sequence is unique for targeting mitochondria. Expression of the gene was repressed in the presence of a phorbol ester in human promyelocyticleukemia cells (HL-60), and this repression was greater than that of the ADP/ATP translocator. These findings suggest that the hinge protein, the expression of which is well regulated, is imported into mitochondria via a specific pathway.