The ⑀ subunit in F 0 F 1 -ATPase/synthase undergoes drastic conformational rearrangement, which involves the transition of two C-terminal helices between a hairpin "down"-state and an extended "up"-state, and the enzyme with the up-fixed ⑀ cannot catalyze ATP hydrolysis but can catalyze ATP synthesis (Tsunoda, S. P.

It has been proposed that C-terminal two ␣-helices of the ⑀ subunit of F 1 -ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F 1 from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the ␥ subunit and C terminus of the ⑀ subunit, respectively. High FRET (extended form) of F 1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5-Adenyl-␤,␥-imidodiphosphate, ADP ؉ AlF 4 ؊ , ADP ؉ NaN 3 , and GTP also caused the retracted form, indicating that ATP binding to the catalytic ␤ subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the ⑀ subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65°C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated ⑀ subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65°C reached to 0.67 mM, a consistent value to assume that the ⑀ subunit acts as a sensor of ATP concentration in the cell.A rotary motor F 1 -ATPase (F 1 ) 2 is a water-soluble portion of F 0 F 1 -ATP synthase, which catalyzes ATP synthesis/hydrolysis coupled with a transmembrane proton translocation (1, 2). F 1 has a subunit structure of ␣ 3 ␤ 3 ␥␦⑀; ␣ and ␤ subunits have a non-catalytic and catalytic nucleotide binding sites, respectively; ␥ subunit rotates in the ␣ 3 ␤ 3 ring; ␦ subunit connects the ring to the stator part of F 0 ; and ⑀ subunit rotates together with ␥ subunit as a body. The ⑀ subunit (ϳ14 kDa) has a regulatory function and consists of N-terminal ␤-sandwitch and C-terminal two ␣-helices (3, 4).Previous structural studies of F 1 indicated two conformations of the ⑀ subunit with different arrangement of the two ␣-helices, that is, retracted folded-hairpin form and partly extended form (Fig. 1, A and B) (5-8). Cross-linking studies suggested the third conformation with fully extended ␣-helices 3 (Fig. 1C) (9). Biochemical data have indicated that the ⑀ subunit adopts the extended form in the absence of nucleotide or in the presence of ADP, in which ATPase activity is inhibited, and that ATP counteracts ADP by favoring the retracted form, which is a noninhibitory conformation (9). Thus, it appears that the regulatory function of the ⑀ subunit is dependent on the drastic conformational transition that is affected by nucleotide and other factors. However, previous studies have not provided kinetic information on how these dynamic conformational transitions occur in the enzyme at work. Fluorescence resonance energy transfer (FRET) is a powerful technique that enables us to probe conformational...

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.Synaptojanins are a family of phosphoinositide phosphatases with a unique three-domain structure: an N-terminal region homologous to yeast Sac1p (1, 2), a central inositol 5-phosphatase domain, and a C-terminal region mediating protein-protein interactions such as Src homology 3 (SH3) 1 domain binding (Ref. 3; for a review, see Ref. 4). The identification and analyses of synaptojanin homologs in lower eukaryotes confirmed that this family of enzymes plays a role of fundamental importance in phosphoinositide metabolism (5-7). Synaptojanin 1, 2 the founding member of this family of enzymes, is a protein predominantly expressed in brain, where it is highly enriched in nerve terminals (8, 9). The C-terminal region of synaptojanin 1 was reported to interact with the SH3 domains of several proteins implicated in the regulation of clathrinmediated synaptic vesicle endocytosis and actin organization at nerve terminals, including amphiphysins (10 -12), endophilins (13,14), intersectins (15), and syndapins (pacsins) (16 -19). These protein-protein interactions are thought to regulate the intracellular localization and activity of synaptojanin 1 (for reviews, see Refs. 20 and 21). The generation and phenotypic analyses of synaptojanin 1-deficient mice (22) and the characterization of Caenorhabditis elegans unc-2...

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Many new biomarkers are being studied, in addition to classical biomarkers, such as chemical substances and their metabolites in blood and urine and modified enzymes. Among these new biomarkers, hemoglobin adducts are thought to be especially useful for the estimation of chemical exposures. We review here the use of biomarkers for monitoring exposures to nine substances, mainly focusing on PRTR class I designated chemical substances, styrene, phenyloxirane (styrene oxide), 4,4'-methylendiphenyl diisocyanate (MDI), 4,4'-methylendianiline (MDA), 1,3-butadiene, ethylene oxide, propylene oxide, acrylamide and acrylonitrile. Hemoglobin adduct levels were elevated after exposures to styrene, MDI, MDA, 1, 3-butadiene, ethylene oxide, acrylamide and acrylonitrile. Moreover, hemoglobin adducts of butadiene, ethylene oxide, acrylamide and acrylonitrile have several useful advantages. For example, the hemoglobin adduct of 1,3-butadiene is an even more useful biomarker of exposure than urinary metabolites, and in the case of ethylene oxide, even though the concentration of ethylene oxide-Hb in the blood of workers did not exceed the value of the German exposure equivalent, a significant difference in it was found between workers and a control group. Also hemoglobin adducts of acrylamide and acrylonitrile can reflect their exposures because there are no urinary metabolites of acrylamide and acrylonitrile that are useful for exposure assessment. In addition to these advantages, hemoglobin adducts are superior to DNA adducts with respect to the availability of large amounts, availability of methods for chemical identification, and well-defined life spans due to the absence of repair. Hemoglobin adducts can be effective biomarkers for assessing exposure to and the effects of chemicals.

We explored the growth of single-walled carbon nanotubes (SWNTs) from nanoparticle array made of an Fe/Al multilayer catalyst by thermal chemical vapor deposition. Fe nanoparticles with a high number density and a narrow size distribution (1-5 nm) were efficiently formed by annealing the Fe/Al thin layer, resulting in the high-yield growth of SWNTs. Moreover, it was found that isolated SWNTs are rooted from patterned Fe/Al islands. The SWNTs bridged between electrodes exhibited semiconducting and metallic properties.

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-Aldehyde dehydrogenase 2 (ALDH2) is an important enzyme that oxidizes acetaldehyde.Approximately 45% of Chinese and Japanese individuals have the inactive ALDH2 genotypes (ALDH2*2/ *2 and ALDH2*1/*2); acute inhalation toxicity of acetaldehyde has not been evaluated in these populations. We compared the toxicity between wild-type (Aldh2+/+) and Aldh2-inactive transgenic (Aldh2−/−) mice by using the paired acute inhalation test modified from the acute toxic class method (OECD TG433). Blood acetaldehyde level was measured 4 hr after the inhalation. A pair of Aldh2+/+ and Aldh2−/− mice was put into a chamber and was exposed to 5000 ppm of acetaldehyde. At the start of the inhalation, the mice exhibited hypoactivity and closing of the eyes. Subsequently, symptoms such as crouching, bradypnea, and piloerection were observed. Flushing was observed only in the Aldh2+/+ mice. Symptoms such as tears, straggling gait, prone position, pale skin, abnormal deep respiration, dyspnea, and one case of death were observed only in the Aldh2−/− mice. The symptoms did not change 1 hr after inhalation in the Aldh2+/+ mice. In contrast, in the Aldh2−/− mice, the symptoms became more severe until the end of the inhalation. The blood acetaldehyde level in the Aldh2−/− mice was approximately twice that in the Aldh2+/+ mice 4 hr after inhalation. The Aldh2−/− mice evidently showed more severe toxicity as compared with the Aldh2+/+ mice due to acute inhalation of acetaldehyde at a concentration of 5000 ppm. Acetaldehyde toxicity in Aldh2+/+ and Aldh2−/− mice was estimated and classified one class different. Based on this study, acetaldehyde inhalations were inferred to pose a higher risk to ALDH2-inactive human individuals.