Purification of N-terminally truncated histone H2A-monoubiquitin conjugates from leukemic cell nuclei: probable proteolytic products of ubiquitinated H2A

To gain insight into the significance of nuclear ubiquitinated proteins, two serial extracts prepared from various leukemic cells were analysed by western blotting with anti-ubiquitin antibody. Two previously unidentified ubiquitinated proteins with molecular masses of 10 and 17 kDa were found in 8 M urea-soluble extracts, obtained from Tris-buffer-insoluble materials, of acute myeloid leukemia OCI/AML 1a cells and the cells from the leukemia patients. Both proteins were successfully purified from the OCI/AML 1a cells and identified as monoubiquitin-truncated H2A conjugates, the 10 kDa ubiquitinated H2A(115-129) and the 17 kDa ubiquitinated H2A(54-129), suggesting that both proteins were produced by limited proteolysis of an intact form (23 kDa) of ubiquitinated H2A(1-129). The 17 kDa protein as well as the 23 kDa ubiquitinated histone H2A were localised in chromatin fractions of the OCI/AML cells and released by high concentrations of salt in a micrococcal nuclease-sensitive manner, suggesting their association with chromatin. In contrast, the 10 kDa protein remained insoluble even when the nuclei were treated with nuclease under high salt concentrations, presumably due to binding to the nuclear matrix. An antibody recognising H2A(70-81) also detected the 17 kDa protein in anti-ubiquitin immunoprecipitates obtained from the OCI/AML cell nuclei. In addition, the 17 kDa protein levels in THP-1 cells were transiently increased, concomitant with a decrease in the 23 kDa ubiquitinated H2A, by treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce differentiation. This is the first report of probable proteolytic products of ubiquitinated H2A, which might have a role in nuclear functions.     

Ostrich antithrombin III: kinetics and mechanism of inhibition of ostrich thrombin

A kinetic investigation of ostrich thrombin specificity, its regulation and evolutionary development in comparison to those of other well-characterised species may contribute to the understanding of the structure-function relationships of thrombin. Antithrombin III (ATIII) was purified from ostrich plasma by heparin-Sepharose and Super Q-650S chromatography. It exhibited a M(r) of 59.2K and a pI in the range of 5.2-6.0. The ostrich N-terminal sequence was compared to those of other known species and showed the highest identity with rabbit ATIII (31%). Inhibition studies included the interaction of ostrich and human ATIII with bovine, human and ostrich thrombin. At a 2:1 molar ratio of ostrich ATIII to enzyme, 20 and 40% remaining activity was found for bovine and ostrich thrombin, respectively. Ostrich thrombin exhibited a pH and temperature optimum of 9.0 and 60 degrees C, respectively. Hydrolysis of seven peptide p-nitroanilide substrates by ostrich thrombin revealed D-Phe-Pip-Arg-pNA (k(cat)/K(m)=9.65 microM(-1)s(-1)) as the substrate with the highest catalytic efficiency. The effect of monovalent cations on ostrich thrombin catalysis revealed enhanced activity with Na(+). The calculated K(i) values for the complex formation between ostrich thrombin and ostrich (9.29 x 10(-11)M) and human (9.66 x 10(-11)M) ATIII are comparable to reported results. The results obtained from the present study confirmed that ostrich thrombin and ATIII are closely related to the corresponding molecules of other species in terms of physicochemical and kinetic properties.     

Tissue generation from amphibian animal caps

Formation of three germ layers is the most important event in early vertebrate development. Animal cap assays can be used to reproduce the in vivo induction of amphibian tissues in order to investigate the differentiation processes that occur in normal embryonic development. Activin treatment strongly and dose-dependently induces various types of mesodermal and endodermal tissue in cultured animal caps. Beating heart, pronephros, pancreas and cartilage can be induced by microsurgical manipulation and simultaneous treatment with activin and other factors. These in vitro induction systems will be helpful for elucidating the mechanisms of tissue induction and organ formation in vertebrate development.     

Requirement of chromatid cohesion proteins rad21/scc1 and mis4/scc2 for normal spindle-kinetochore interaction in fission yeast

BACKGROUND: Proteins conserved from yeast to human hold two sister chromatids together. The failure to form cohesion in the S phase results in premature separation of chromatids in G2/M. Mitotic kinetochores free from microtubules or the lack of tension are known to activate spindle checkpoint. RESULTS: The loss of chromatid cohesion in fission yeast mutants (mis4-242 and rad21-K1) leads to the activation of Mad2- and Bub1-dependent checkpoint, possibly due to a diminished microtubule-kinetochore interaction. Bub1, a checkpoint kinase, localizes briefly at early mitotic kinetochores in wild-type, whereas the cohesion mutation greatly increases the duration of kinetochore localization. Bub1 is bound to the central centromere region of mitotic cells. These cohesion mutants are hypersensitive to a tubulin poison and are synthetic lethal with dis1 and bir1/cut17, which are defective in microtubule-kinetochore interaction. The formation of specialized centromere chromatin containing CENP-A does not require cohesion. Dominant-negative noncleavable Rad21 fails to activate checkpoint but blocks sister chromatid separation and full spindle elongation in anaphase. CONCLUSIONS: Mis4 and Rad21 (budding yeast Scc2 and Scc1 homologs, respectively) act in establishing the normal spindle-kinetochore interaction in early mitosis and inhibit sister chromatid separation until the cleavage of Rad21 in anaphase. Checkpoint directly or indirectly monitors the states of cohesion in early mitosis. Full spindle extension occurs with unequal nuclear division in cohesion mutants in the absence of Mad2.     

Enhancement of Ethanol Fermentation in Saccharomyces cerevisiae Sake Yeast by Disrupting Mitophagy Function

Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 has one of the highest fermentation rates among brewery yeasts used worldwide; therefore, it is assumed that it is not possible to enhance its fermentation rate. However, in this study, we found that fermentation by sake yeast can be enhanced by inhibiting mitophagy. We observed mitophagy in wild-type sake yeast during the brewing of Ginjo sake, but not when the mitophagy gene (ATG32) was disrupted. During sake brewing, the maximum rate of CO₂ production and final ethanol concentration generated by the atg32Δ laboratory yeast mutant were 7.50% and 2.12% higher than those of the parent strain, respectively. This mutant exhibited an improved fermentation profile when cultured under limiting nutrient concentrations such as those used during Ginjo sake brewing as well as in minimal synthetic medium. The mutant produced ethanol at a concentration that was 2.76% higher than the parent strain, which has significant implications for industrial bioethanol production. The ethanol yield of the atg32Δ mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that the atg32Δ mutant has acquired a high fermentation capability at the cost of decreasing biomass. Because natural biomass resources often lack sufficient nutrient levels for optimal fermentation, mitophagy may serve as an important target for improving the fermentative capacity of brewery yeasts.     

Effects of Resistance Exercise on Iron Absorption and Balance in Iron-Deficient Rats

We have previously reported that resistance exercise improved the iron status in iron-deficient rats. The current study investigated the mechanisms underlying this exercise-related effect. Male 4-week-old rats were divided into a group sacrificed at the start (week 0) (n?=?7), a group maintained sedentary for 6 weeks (S) or a group that performed exercise for 6 weeks (E), and all rats in the latter groups were fed an iron-deficient diet (12 mg iron/kg) for 6 weeks. The rats in the E group performed climbing exercise (5 min?×?6 sets/day, 3 days/week). Compared to the week 0 rats, the rats in the S and E groups showed lower tissue iron content, and the hematocrit, hemoglobin, plasma iron, and transferrin saturation values were all low. However, the tissue iron content and blood iron status parameters, and the whole body iron content measured using the whole body homogenates of the rats, did not differ between the S group and the E group. The messenger RNA (mRNA) expression levels of hepcidin, duodenal cytochrome b, divalent metal transporter 1, and ferroportin 1 did not differ between the S group and the E group. The apparent absorption of iron was significantly lower in the E group than in the S group. Therefore, it was concluded that resistance exercise decreases iron absorption, whereas the whole body iron content is not affected, and an increase in iron recycling in the body seems to be responsible for this effect.     

Molecular interactions of the quinone electron acceptors QA,QB, and QC in photosystem II as studied by the fragment molecular orbital method

Molecular interactions of the three plastoquinone electron acceptors, Q_A, Q_B, and Q_C, in photosystem II (PSII) were studied by fragment molecular orbital (FMO) calculations. Calculations at the FMO-MP2/6-31G level using PSII models deduced from the X-ray structure of the PSII complexes from Thermosynechococcus elongatus provided the binding energies of Q_A, Q_B, and Q_C as ?56.1, ?37.9, and ?30.1 kcal/mol, respectively. The interaction energies with surrounding fragments showed that the contributions of lipids and cofactors were 0, 24 and 45 % of the total interaction energies for Q_A, Q_B, and Q_C, respectively. These results are consistent with the fact that Q_A is strongly bound to the PSII protein, whereas Q_B functions as a substrate and is exchangeable with other quinones and herbicides, and the presence of Q_C is highly dependent on PSII preparations. It was further shown that the isoprenoid tail is more responsible for the binding than the head group in all the three quinones, and that dispersion forces rather than electrostatic interactions mainly contribute to the stabilization. The relevance of the stability and molecular interactions of Q_A, Q_B, and Q_C to their physiological functions is discussed.     

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to d-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2′-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.     

Role of coarse woody debris in the carbon cycle of Takayama forest,central Japan

Coarse woody debris (CWD) is an important component of the forest carbon cycle, acting as a carbon pool and a source of CO₂ in temperate forest ecosystems. We used a soda-lime closed-chamber method to measure CO₂ efflux from downed CWD (diameter ≥5 cm) and to examine CWD respiration (R _CWD) under field conditions over 1 year in a temperate secondary pioneer forest in Takayama forest. We also investigated tree mortality (input to the CWD pool) from the data obtained from the annual tree census, which commenced in 2000. We developed an exponential function of temperature to predict R _CWD in each decay class (R ² = 0.81–0.97). The sensitivity of R _CWD to changing temperature, expressed as Q ₁₀, ranged from 2.12 to 2.92 and was relatively high in decay class III. Annual C flux from CWD (F _CWD) was extrapolated using continuous air temperature measurements and CWD necromass pools in the three decay classes. F _CWD was 3.0 (class I), 17.8 (class II), and 13.7 g C m^?2 year^?1 (class III) and totaled 34 g C m^?2 year^?1 in 2009. Annual input to CWD averaged 77 g C m^?2 year^?1 from 2000 to 2009. The budget of the CWD pool in the Takayama forest, including tree mortality inputs and respiratory outputs, was 0.43 Mg C ha^?1 year^?1 (net C sink) owing to high tree mortality in the mature pioneer forest. The potential CWD sink is important for the carbon cycle in temperate successional forests.