Purification and Characterization of DNA Polymerase Obtained from the Membrane Fraction of E. coli

Purification studies were conducted on DNA polymerase bound to the membrane fraction of E. coli HF 4704. Purified enzyme (Fraction V) required Mg²⁺ and showed an optimun pH of 7.2. Various kinds of salt indicated a stimulative effect at concentrations lower than 0.1 m. Fraction V was unstable at an acidic condition (pH 5.0) but was rather stable at an alkaline condition (pH 9.0). The enzyme activity was lost by incubation at 45°C for 30min but was stabilized by the addition of DNA. The enzyme contained exonuclease activity but no endonuclease activity. The enzyme produced only light density DNA of various sizes. The function of this enzyme as considered to fill single stranded region of the double stranded primer DNA.     

BAT1, a putative acyltransferase,modulates brassinosteroid levels in Arabidopsis

Brassinosteroids (BRs) are essential for various aspects of plant development. Cellular BR homeostasis is critical for proper growth and development of plants; however, its regulatory mechanism remains largely unknown. BAT1 (BR‐related acyltransferase 1), a gene encoding a putative acyltransferase, was found to be involved in vascular bundle development in a full‐length cDNA over‐expressor (FOX) screen. Over‐expression of BAT1 resulted in typical BR‐deficient phenotypes, which were rescued by exogenously applied castasterone and brassinolide. Analyses of BR profiles demonstrated that BAT1 alters levels of several brassinolide biosynthetic intermediates, including 6‐deoxotyphasterol, typhasterol and 6‐deoxocastasterone. BAT1 is mainly localized in the endoplasmic reticulum. BAT1 is highly expressed in young tissues and vascular bundles, and its expression is induced by auxin. These data suggest that BAT1 is involved in BR homeostasis, probably by conversion of brassinolide intermediates into acylated BR conjugates.     

Discovery of novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones as human chymase inhibitors using structure-based drug design

A novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones were explored as human chymase inhibitors using structure-based drug design according to the X-ray cocrystal structure of chymase and compound 1. The optimization focused on the prime site led to the attainment of compounds that showed potent inhibitory activity, and among them, 18R shows a novel interaction mode.     

Effect of Resistance Exercise on Iron Status in Moderately Iron-Deficient Rats

Resistance exercise increases heme synthesis in the bone marrow, but it does not improve the hemoglobin status in severe iron-deficient rats on a diet containing less than 5?mg iron/kg. The current study investigated whether resistance exercise could mitigate hemoglobin status via increasing heme synthesis in moderately iron-deficient rats. Male 4-week-old Sprague-Dawley rats were fed an iron-deficient diet containing 12?mg iron/kg for 3?weeks. The rats were divided into two groups: a sedentary (S) group (n?=?7) or an exercise (E) group (n?=?7). The rats in the E group performed a climbing exercise (5?min?×?6?sets/day, 3?days/week). The aminolevulinic acid dehydratase activity, hematocrit, and hemoglobin tended to be higher in group E than S. The iron content in the flexor hallucis longus muscle was significantly higher in E than S, whereas the content in the liver, spleen, kidney, and heart did not significantly differ between the groups. Therefore, resistance exercise appears to improve hemoglobin via increasing heme synthesis in the bone marrow in moderately iron-deficient rats.     

Element Distribution in Visual System,the Optic Chiasma,Lateral Geniculate Body,and Superior Colliculus

To elucidate compositional changes of the visual system with aging, the authors investigated age-related changes of elements in the optic chiasma, lateral geniculate body, and superior colliculus, relationships among their elements, relationships among their brain regions from a viewpoint of elements, and gender differences in their elements by direct chemical analysis. After ordinary dissection at Nara Medical University was finished, the optic chiasmas, lateral geniculate bodies, and superior colliculi were resected from identical cerebra of the subjects. The subjects consisted of 14 men and 10 women, ranging in age from 75 to 96 years (average age = 85.6 ± 5.9 years). After ashing with nitric acid and perchloric acid, element contents were determined by inductively coupled plasma-atomic emission spectrometry. As the result, the average content of P was significantly higher in the optic chiasma and superior colliculus compared with the lateral geniculate body. Regarding age-related changes of elements, no significant changes with aging were found in seven elements of the optic chiasma, lateral geniculate body, and superior colliculus in the subjects more than 75 years of age. The findings that with regard to the relationships among elements, there were extremely significant direct correlations between Ca and Zn contents and significant inverse correlations between Mg and Na contents were obtained in common in all of the optic chiasma, lateral geniculate body, and superior colliculus. It was examined whether there were significant correlations among the optic chiasma, lateral geniculate body, and superior colliculus in the seven elements and the following results were obtained: There were significant direct correlations between the optic chiasma and lateral geniculate body in both the P and Mg contents; there was a significant direct correlation between the optic chiasma and superior colliculus in the Fe content; and a significant direct correlation was found between the lateral geniculate body and superior colliculus in the Mg content. Regarding the gender differences in elements, it was found that both the Ca and Zn contents of the lateral geniculate body were significantly higher in women than in men.     

Transfer learning for cytochrome P450 isozyme selectivity prediction

In the drug discovery process, the metabolic fate of drugs is crucially important to prevent drug-drug interactions. Therefore, P450 isozyme selectivity prediction is an important task for screening drugs of appropriate metabolism profiles. Recently, large-scale activity data of five P450 isozymes (CYP1A2 CYP2C9, CYP3A4, CYP2D6, and CYP2C19) have been obtained using quantitative high-throughput screening with a bioluminescence assay. Although some isozymes share similar selectivities, conventional supervised learning algorithms independently learn a prediction model from each P450 isozyme. They are unable to exploit the other P450 isozyme activity data to improve the predictive performance of each P450 isozyme's selectivity. To address this issue, we apply transfer learning that uses activity data of the other isozymes to learn a prediction model from multiple P450 isozymes. After using the large-scale P450 isozyme selectivity dataset for five P450 isozymes, we evaluate the model's predictive performance. Experimental results show that, overall, our algorithm outperforms conventional supervised learning algorithms such as support vector machine (SVM), Weighted k-nearest neighbor classifier, Bagging, Adaboost, and latent semantic indexing (LSI). Moreover, our results show that the predictive performance of our algorithm is improved by exploiting the multiple P450 isozyme activity data in the learning process. Our algorithm can be an effective tool for P450 selectivity prediction for new chemical entities using multiple P450 isozyme activity data.     

Vital role of the calpain-calpastatin system for placental-integrity-dependent embryonic survival

Although the calpain-calpastatin system has been implicated in a number of pathological conditions, its normal physiological role remains largely unknown. To investigate the functions of this system, we generated conventional and conditional calpain-2 knockout mice. The conventional calpain-2 knockout embryos died around embryonic day 15, preceded by cell death associated with caspase activation and DNA fragmentation in placental trophoblasts. In contrast, conditional knockout mice in which calpain-2 is expressed in the placenta but not in the fetus were spared. These results suggest that calpain-2 contributes to trophoblast survival via suppression of caspase activation. Double-knockout mice also deficient in calpain-1 and calpastatin resulted in accelerated and rescued embryonic lethality, respectively, suggesting that calpain-1 and -2 at least in part share similar in vivo functions under the control of calpastatin. Triple-knockout mice exhibited early embryonic lethality, a finding consistent with the notion that this protease system is vital for embryonic survival.     

Ubiquitination and downregulation of ErbB2 and estrogen receptor-alpha by kinase inhibitor MP-412 in human breast cancer cells

ErbB2 has been proven to be an important target for breast cancer therapy. MP-412 is a dual ErbB2 and epidermal growth factor receptor tyrosine kinase inhibitor belonging to an irreversible-type anilinoquinazoline derivative. We demonstrate herein that along with the kinase inhibition, MP-412 has the ability to induce ubiquitination, internalization, and degradation of ErbB2 in several human breast cancer cell lines at concentrations relatively higher than those required for kinase inhibition. Another irreversible inhibitor, CI-1033, showed similar activity, while the reversible compounds were ineffective, suggesting a crucial role of covalent bonding functionality in these effects. In MCF7 cells, MP-412 depleted not only ErbB2 but also estrogen receptor (ER)-α, and to some extent, affected Raf-1, while MP-412 activated Hsp70 expression. Moreover, we observed that MP-412 increased immunocomplexing of Hsp70 with ErbB2 and ER-α, with simultaneous induction of ubiquitination of these client proteins. Furthermore, in combination with proteasome inhibitor, MP-412 resulted in the noticeable accumulation of ErbB2 and ER-α in the detergent insoluble fraction of cell lysates. These results suggest that MP-412 acts as an inhibitor of Hsp90 function, whereas MP-412 did not bind directly to ATP-binding site of Hsp90, unlike geldanamycin. We also found that new protein synthesis was involved in the activity of MP-412 on Hsp90 modulation. Since downregulation of ErbB2 and ER-α by accelerating the ubiquitin-proteolysis system will become an attractive approach for breast cancer therapy, we expect MP-412 to be a lead compound for the drug design and the development of such agents.     

A reduction of licensed origins reveals strain-specific replication dynamics in mice

Replication origin licensing builds a fundamental basis for DNA replication in all eukaryotes. This occurs during the late M to early G1 phases in which chromatin is licensed by loading of the MCM2-7 complex, an essential component of the replicative helicase. In the following S phase, only a minor fraction of chromatin-bound MCM2-7 complexes are activated to unwind the DNA. Therefore, it is proposed that the vast majority of MCM2-7 complexes license dormant origins that can be used as backups. Consistent with this idea, it has been repeatedly demonstrated that a reduction (~60%) in chromatin-bound MCM2-7 complexes has little effect on the density of active origins. In this study, however, we describe the first exception to this observation. A reduction of licensed origins due to Mcm4 ^chaos3 homozygosity reduces active origin density in primary embryonic fibroblasts (MEFs) in a C57BL/6J (B6) background. We found that this is associated with an intrinsically lower level of active origins in this background compared to others. B6 Mcm4 ^{chaos3/chaos3} cells proliferate slowly due to p53-dependent upregulation of p21. In fact, the development of B6 Mcm4 ^{chaos3/chaos3} mice is impaired and a significant fraction of them die at birth. While inactivation of p53 restores proliferation in B6 Mcm4 ^{chaos3/chaos3} MEFs, it paradoxically does not rescue animal lethality. These findings indicate that a reduction of licensed origins may cause a more profound effect on cell types with lower densities of active origins. Moreover, p53 is required for the development of mice that suffer from intrinsic replication stress.     

β-Catenin is essential for Müllerian duct regression during male sexual differentiation

During male sexual differentiation, the transforming growth factor-β (TGF-β) signaling molecule anti-Müllerian hormone (AMH; also known as Müllerian inhibiting substance, MIS) is secreted by the fetal testes and induces regression of the Müllerian ducts, the primordia of the female reproductive tract organs. Currently, the molecular identity of downstream events regulated by the AMH signaling pathway remains unclear. We found that male-specific Wnt4 expression in mouse Müllerian duct mesenchyme depends upon AMH signaling, implicating the WNT pathway as a downstream mediator of Müllerian duct regression. Inactivation of β-catenin, a mediator of the canonical WNT pathway, did not affect AMH signaling activation in the Müllerian duct mesenchyme, but did block Müllerian duct regression. These data suggest that β-catenin mediates AMH signaling for Müllerian duct regression during male sexual differentiation.