N-(6-phenylpyridazin-3-yl)benzenesulfonamides as highly potent, brain-permeable, and orally active kynurenine monooxygenase inhibitors.

Huntington's disease (HD) is one of the serious neurodegenerative diseases and no disease modifiers are available to date. The correction of unbalanced kynurenine pathway metabolites may be useful to treat disease progression and kynurenine monooxygenase (KMO) is considered an ideal drug target. A couple of KMO inhibitors have been reported, but their brain permeability was very poor. We found pyridazinylsulfonamide as a novel lead compound, and it was optimized to the brain-permeable and highly potent KMO inhibitor 12, which was equipotent with CHDI-340246 and superior to CHDI-340246 in terms of brain penetration. Compound 12 was effective in R6/2 mice (HD model mice), i.e. neuroprotective kynurenic acid was increased, whereas neurotoxic 3-hydroxykynurenine was suppressed. In addition, impaired cognitive function was improved. Therefore, the brain-permeable KMO inhibitor was considered to be a disease modifier for HD treatment.

Impaired p53/CEP-1 is associated with lifespan extension through an age-related imbalance in the energy metabolism of C. elegans.

In the nematode Caenorhabditis elegans, the mammalian tumor suppressor p53 ortholog CEP-1 mediates the stress response, activates germ line apoptosis and regulates meiotic chromosome segregation. A reduction in its expression, which frequently occurs in mammalian cancer cells, extends lifespan and induces an adaptive response in C. elegans. However, these effects do not involve an increase in oxidative stress resistance. Here, we showed that intermittent exposure to hyperoxia, which induces oxidative stress resistance and lowers the production of ROS derived from mitochondrial respiration in C. elegans, slightly improved the lifespan extension of cep-1 mutant. Interestingly, ATP levels were increased without an increase in oxygen consumption in cep-1 mutant during aging. In the wild-type, lactate levels and consequentially the lactate/pyruvate ratio decreased during aging in adults. Furthermore, the expression levels of mitochondrial respiration-related sco-1, which is a target of p53/CEP-1, as well as those of gluconeogenesis regulation and mammalian sirtuin ortholog genes, were also increased in the aged and adaptive conditioned wild-type animals. In contrast, the lactate/pyruvate ratio increased in cells of the cep-1 mutant and was amplified by intermittent hyperoxia. These results suggest that impaired p53/CEP-1 leads to an imbalance in the age-related energy metabolic alteration between mitochondrial oxidative phosphorylation and aerobic glycolysis and plays an important role in the extension of both intact and adaptive lifespans.

Biophysical and biological meanings of healthspan from C. elegans cohort.

Lifespan among individuals ranges widely in organisms from yeast to mammals, even in an isogenic cohort born in a nearly uniform environment. Needless to say, genetic and environmental factors are essential for aging and lifespan, but in addition, a third factor or the existence of a stochastic element must be reflected in aging and lifespan. An essential point is that lifespan or aging is an unpredictable phenomenon. The present study focuses on elucidating the biophysical and biological meanings of healthspan that latently indwells a stochastic nature. To perform this purpose, the nematode Caenorhabditis elegans served as a model animal. C. elegans fed a healthy food had an extended healthspan as compared to those fed a conventional diet. Then, utilizing this phenomenon, we clarified a mechanism of healthspan extension by measuring the single-worm ATP and estimating the ATP noise (or the variability of the ATP content) among individual worms and by quantitatively analyzing biodemographic data with the lifespan equation that was derived from a fluctuation theory.

Noise-driven onset time of biodemographic aging.

The lifespan of each individual, even in an isogenic cohort and a uniform environment, is quite different. The genetic factors influencing the lifespan in humans as well as animal models are few. The balance is attributed to "chance" variations. In this study, we focus on a third factor, noise or chance variations, as well as on genetic and environmental factors and examine how biodemographic aging is related to stochastic fluctuations, or noise. To elucidate the third factor in relation to aging and lifespan, we employed the nematode Caenorhabditis elegans, which can provide an ideal system for analyzing the mathematical and biophysical models. An amplification of ATP noise was clearly evident from around the onset of biodemographic aging (t(0)) as if the t(0) was synchronized with or derived from the amplification of noise. Furthermore, the expression noise of the unc-54 gene, which encodes the myosin heavy chain, increased from around the t(0). In contrast, the noise of genes related to the mitochondrial respiratory chain was almost constant with aging. There is a high energy barrier between life and death. Here we propose that the transition from living to dying may be facilitated by noise amplification. The finite value (or non-zero) of t(0) is essential to the lifespan equation derived from the diffusion model.

Timing mechanism and effective activation energy concerned with aging and lifespan in the long-lived and thermosensory mutants of Caenorhabditis elegans.

The lifespans of many poikilothermic animals, including the nematode Caenorhabditis elegans, depend significantly on environmental temperature. Using long-living, thermosensory mutants of C. elegans, we tested whether the temperature dependency of the mean lifespan is compatible with the Arrhenius equation, which typically represents one of the chemical reaction rate theories. The temperature dependency of C. elegans was the Arrhenius type or normal, but daf-2(e1370) mutants were quite different from the others. However, taking into account the effect of the thermal denaturation of DAF-2 with the temperature, we showed that our analyzed results are compatible with previous ones. We investigated the timing mechanism of one parameter (the onset of biodemographic aging (t(0))) in the lifespan equation by applying the RNAi feeding method to daf-2 mutants in order to suppress daf-16 activity at different times during the life cycle. In summary, we further deepened the biological role of two elements, t(0) and z (the inverse of the aging rate), in the lifespan equation and mean lifespan formulated by our diffusion model z(2) = 4Dt(0), where z is composed of t(0) and D (the diffusion constant).

A further test of the equation of lifespan by C. elegans: effective activation energy for aging and lifespan.

We previously proposed a rate theory of chemical reaction as well as a lifespan equation derived by a stochastic fluctuation theory. Both were applied to biodemographic data by C. elegans to quantitatively explain that respiratory activity declines exponentially with age and that it has a physiological decline rate and a finite value (threshold) in advanced age. In this work, using the poikilothermic nature of Caenorhabditis elegans, we demonstrate the further validity of the rate theory of chemical reaction as well as the lifespan equation by changing two methods. First, to test the appropriateness of the lifespan equation from another aspect, lifespan assays were conducted by varying the time interval of observation employing the egl-1 mutant. The results indicate that, as the time interval is reduced, mortality rates gradually approach the force of mortality expected from the fitting equation of the survival curve. Second, based on the dependence of lifespan on the temperature of the culture, the physiological decline rate, and the onset of biodemographic aging, we show that the effective activation energy or energy barrier for aging and lifespan may be closely related to the standard free-energy change of ATP or ADP for a wild type and some lifespan-related mutants of C. elegans.

Interrelationships between mitochondrial fusion, energy metabolism and oxidative stress during development in Caenorhabditis elegans.

Mitochondria are known to be dynamic structures with the energetically and enzymatically mediated processes of fusion and fission responsible for maintaining a constant flux. Mitochondria also play a role of reactive oxygen species production as a byproduct of energy metabolism. In the current study, interrelationships between mitochondrial fusion, energy metabolism and oxidative stress on development were explored using a fzo-1 mutant defective in the fusion process and a mev-1 mutant overproducing superoxide from mitochondrial electron transport complex II of Caenorhabditis elegans. While growth and development of both single mutants was slightly delayed relative to the wild type, the fzo-1;mev-1 double mutant experienced considerable delay. Oxygen sensitivity during larval development, superoxide production and carbonyl protein accumulation of the fzo-1 mutant were similar to wild type. fzo-1 animals had significantly lower metabolism than did N2 and mev-1. These data indicate that mitochondrial fusion can profoundly affect energy metabolism and development.

Decline in oxygen consumption correlates with lifespan in long-lived and short-lived mutants of Caenorhabditis elegans.

In humans, the basal energy metabolism is thought to decline linearly with age. On the other hand, in the nematode Caenorhabditis elegans, two research groups reported independently that it declined exponentially. In this study, furthermore, we used various lifespan-mutant strains to determine whether the previous conclusion is more likely to be true. We can indirectly estimate the metabolic energy by conveniently measuring the oxygen consumption rates of C. elegans using an optical apparatus. From the profile of respiratory rates as a function of age, we can quantitatively isolate the physiological decline rate, lambda, that exponentially represents the decay rate of respiratory activity with age. In addition, quantitative analysis indicates that the respiratory activity of worms has a finite value in advanced age. We also show that the maximum and mean lifespans strongly correlate with the reciprocal of the lambda. These findings offer crucial biochemical evidence for a molecular mechanism at work in biological aging. Consequently, we here propose a mechanism based on a chemical reaction and offer a definition of the physiological decline rate and the finiteness of respiratory activity in advanced age.

Analyzing observed or hidden heterogeneity on survival and mortality in an isogenic C. elegans cohort.

It is generally difficult to understand the rates of human mortality from biological and biophysical standpoints because there are no cohorts or genetic homogeneity; in addition, information is limited regarding the various causes of death, such as the types of accidents and diseases. Despite such complexity, Gompertz's rule is useful in humans. Thus, to characterize the rates of mortality from a demographic viewpoint, it would be interesting to research a single disease in one of the simplest organisms, the nematode C. elegans, which dies naturally under identically controlled circumstances without predators. Here, we report an example of the fact that heterogeneity on survival and mortality is observed through a single disease in a cohort of 100% genetically identical (isogenic) nematodes. Under the observed heterogeneity, we show that the diffusion theory, as a biophysical model, can precisely analyze the heterogeneity and conveniently estimate the degree of penetrance of a lifespan gene from the biodemographic data. In addition, we indicate that heterogeneity models are effective for the present heterogeneous data.

Basic principle of the lifespan in the nematode C. elegans.

We present a biophysical model based on the principles of fluctuation and regulation to explain the effect of stochastics on survival. The model is a good fit for the survivorship and mortality rates observed in the nematode Caenorhabditis elegans. A parameter included in the theory, which is called the fluctuation constant, correlates well with a change (or declining rate) of respiration with age, which we term the physiological decline rate. The square of the physiological decline rate is proportional to the reciprocal of the fluctuation constant as revealed in a diffusion equation. In addition, the maximum and mean life spans are proportional to the reciprocal of the decline rate. The framework involved in the fluctuation theory is compatible with the existence of a regulatory system such as that acting in the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway during adulthood, and that sensing, switching, and memorizing the rate of mitochondrial respiration early in life.

Analysis of mismatched DNA by mismatch binding ligand (MBL)-Sepharose affinity chromatography.

Mismatch binding molecules (MBLs), strongly and selectively bound to the mismatched base pair in duplex DNA, were immobilized on Sepharose. Three MBL-Sepharose columns were prepared with three MBLs, naphthyridine dimer (ND), naphthyridine-azaquinolone (NA), and aminonaphthyridine dimer (amND), which exhibited different binding profiles to the mismatched base pairs. These three MBL-Sepharose columns showed characteristic elution profiles for DNA duplexes containing mismatched base pairs. The ND-Sepharose column separated the G-G and G-A mismatched DNA from fully matched duplexes. The NA-Sepharose column separated the A-A and G-A mismatched DNA from other DNA duplexes. The amND-Sepharose column separated the C-C mismatched DNA. These chromatographic profiles were very consistent with the binding preference of each MBL. By changing the elution conditions from sodium hydroxide to sodium chloride, MBL-Sepharose columns were also able to separate the mismatched DNA that weakly bound to the MBL from fully matched DNA duplex. Figure MBL-Sepharose affinity chromatography successfully separates the mismatched duplex DNA from fully matched duplex.

Allele specific C-bulge probes with one unique fluorescent molecule discriminate the single nucleotide polymorphism in DNA.

A combination of an allele specific C-bulge probe and the fluorescent molecule N,N'-bis(3-aminopropyl)-2,7-diamino-1,8-naphthyridine (DANP) that binds specifically to the C-bulge provides a method for single nucleotide polymorphism (SNP) typing with only one fluorescent molecule without covalent modification of the DNA probe. The allele specific C-bulge probe contains one additional cytosine and produces a C-bulge directly flanking the SNP site upon hybridization to the target DNA. The C-bulge is a scaffold to recruit and retain DANP directly neighboring the SNP site. The DANP fluorescent probe was selectively modulated by the flanking matched and mismatched base pairs. The mutation type could be discriminated by the modulated fluorescent intensity with respect to the allele specific C-bulge probes used for the assay.

Evidence for the existence of a soybean resistant protein that captures bile acid and stimulates its fecal excretion.

Feeding HMF, an insoluble "high-molecular-weight fraction" from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestible protein or peptide that can be called a "resistant protein" in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestible fraction of HMF.

Age-related changes of mitochondrial structure and function in Caenorhabditis elegans.

A number of observations have been made to examine the role that mitochrondrial energetics and superoxide anion production play in the aging of wild-type Caenorhabditis elegans. Ultrastructural analyses reveal the presence of swollen mitochondria, presumably produced by fusion events. Two key mitochondrial functions - the activity of two electron transport chain complexes and oxygen consumption - decreased as animals aged. Carbonylated proteins, one byproduct of oxidative stress, accumulated in mitochondria much more than in the cytoplasm. This is consistent with the notion that mitochondria are the primary source of endogenous reactive oxygen species. However, the level of mitochondrially generated superoxide anion did not change significantly during aging, suggesting that the accumulation of oxidative damage is not due to excessive production of superoxide anion in geriatric animals. In concert, these data support the notion that the mitochondrial function is an important aging determinant in wild-type C. elegans.

N,N'-Bis(3-aminopropyl)-2,7-diamino-1,8-naphthyridine stabilized a single pyrimidine bulge in duplex DNA.

We here show the first identified ligand 2,7-diamino-1,8-naphthyridine (DANP) that strongly and specifically binds to the single cytosine and thymine bulges with exclusively 1:1 stoichiometry.

Direct measurement of oxygen consumption rate on the nematode Caenorhabditis elegans by using an optical technique.

It is well known that aging and longevity strongly correlate with energy metabolism. The nematode Caenorhabditis elegans is widely used as an ultimate model of experimental animals. Thus, we developed a novel tool, which is constructed from an optical detector, using an indirect method that can measure simply the energy metabolism of C. elegans. If we measure the oxygen consumption rate using this optical tool, we can easily evaluate the activity of mitochondria as an index in the aging process. However, a direct measurement of the oxygen consumption rate of C. elegans exposed in air is thought to be impossible because of the high concentration of atmospheric oxygen and the small size of the animals. We demonstrate here that we can directly detect the oxygen consumption with a small number of animals (

2-Ureidoquinoline: a useful molecular element for stabilizing single cytosine and thymine bulges.

We have demonstrated that aromatic heterocycles having hydrogen-bonding surfaces complementary to those of nucleotide bases are effective molecular elements for the binding to single nucleotide bulges and base mismatches. We here report that a new molecule, 2-ureidoquinoline having an alignment of hydrogen-bonding groups in the order of acceptor-donor-donor stabilizes single cytosine and thymine bulges in duplex DNAs. Furthermore, a dimeric form of 2-ureidoquinoline stabilizes cytosine-cytosine and cytosine-thymine mismatches.

Force generation by recombinant myosin heads trapped between two functionalized surfaces.

Fluorescence resonance energy transfer measurements have revealed that the lever-arm domain of myosin swings when it hydrolyzes Mg-ATP. It is generally accepted that this swing of the lever arm of myosin is the molecular basis of force generation. On the other hand, the possibility that the force might be generated at the interface between actin and myosin cannot be ignored. However, there is a third possibility, namely, that myosin itself generates force without actin. Thus, using recombinant subfragment 1 molecules of Dictyostelium myosin II that were trapped between two functionalized surfaces of a surface-force apparatus, we determined whether myosin itself could actually generate force. Here, we report that, despite the absence of actin, myosin heads themselves have a capacity to generate a force (at least approximately 0.2 pN/molecule) that is coupled to the structural changes. Although the role of actin should not be neglected because muscle physiologically shortens as a result of the interaction between actin and myosin, in this work the focus is on the question of whether the catalytic domain of myosin has the capacity to generate force.

The SPR sensor detecting cytosine[bond]cytosine mismatches.

We have synthesized the first surface plasmon resonance (SPR) sensor that detects cytosine-cytosine (C[bond]C) mismatches in duplex DNA by immobilizing aminonaphthyridine dimer on the gold surface. The ligand consisting of two 2-aminonaphthyridine chromophores and an alkyl linker connecting them strongly stabilized the C[bond]C mismatches regardless of the flanking sequences. The fully matched duplexes were not stabilized at all under the same conditions. The C[bond]T, C[bond]A, and T[bond]T mismatches were also stabilized with a reduced efficiency. SPR analyses of mismatch-containing 27-mer duplexes were performed with the sensor surface on which the aminonaphthyridine dimer was immobilized. The response for the C[bond]C mismatch in 5'-GCC-3'/3'-CCG-5' was about 83 times stronger than that obtained for the fully matched duplex. The sensor successfully detects the C[bond]C mismatch at the concentration of 10 nM. SPR responses are proportional to the concentration of the C[bond]C mismatch in a range up to 200 nM. Aminonaphthyridine dimer could bind strongly to the C[bond]C mismatches having 10 possible flanking sequences with association constants in the order of 10(6) M(-1). The facile protonation of 2-aminonaphthyridine chromophore at pH 7 producing the hydrogen-bonding surface complementary to that of cytosine was most likely due to the remarkably high selectivity of 1 to the C[bond]C mismatch.

Separation of mismatched DNA by using the affinity column immobilizing mismatch-binding ligands.

Mismatch binding ligands (MBL), ND and NA, bind to DNA containing mismatch base pairs. These MBLs were immobilized to NHS-activated affinity column via amine linker. Affinity chromatographic analyses of mismatched DNAs by using the ND-immobilized column showed clear separation of G-G and G-A from other mismatched DNAs. The results of the NA-immobilized column also showed good separation of A-A, G-A, G-G, A-C from other mismatched DNAs. The order of mobility of mismatched duplexes on MBL-immobilized column was in good agreement with that of the binding affinity in a solution phase obtained by the melting temperature measurement.