Search

Potential toxic effects of CWP232228 in nude mice.

Sphere-forming 4T1 cell culture leads to the expression of stem cell markers.

The comparison of CWP232228 activities with another well-known small molecule inhibitor.

Supplementary figure legends

Pharmacokinetic/pharmacodynamic (PK/PD) analysis.

Characterization of primary human breast cancer cells.

Sphere-forming MDA-MD-435 cell culture leads to the expression of stem cell markers.

Gene expression profiles of breast cancer cells grown as monolayer or sphere-forming cells.

The expression levels of stem cell markers.

The extreme limiting dilution(ELDA)assay.

Chemical structure and pharmacokinetic properties of the small-molecular weight inhibitor CWP232228.

The list of the PCR primer sequences.

Expression profiles of the genes associated with Wnt/β-catenin signaling in premalignant mammary and breast cancer tissues.

Effect of CWP232228 on the growth of BCSCs obtained from human primary breast cancers.

The behavior of nuclear structures with an increase in atomic number from $${}^{150}$$ Nd to $${}^{158}$$ Er was investigated in this study. $${}^{152}$$ Sm and $${}^{154}$$ Gd are typical nuclei lying between the rotational and vibrational states, which can be explained by a slight breaking of the SU(3) symmetry in the U(5)-direction via the interacting boson model (IBM). Furthermore, $$N=90$$ isotones lie in the path of this symmetry-breaking phase transition. Moreover, the nuclear structure of $${}^{150}$$ Nd can be explained using X(5) symmetry. However, because the $${}^{156}$$ Dy and $${}^{158}$$ Er nuclei are not fully symmetrical, they can be represented by adding a perturbed term to express symmetry breaking. We identified the tendency of change in the nuclear structure using the following three calculation steps. First, the structures of $${}^{152}$$ Sm and $${}^{154}$$ Gd were described using the matrix elements of the Hamiltonian and the electric quadrupole operator between the basis states of the SU(3) limit in the IBM. Second, the low-lying energy levels and E2 transition ratios corresponding to the observable physical values were calculated by adding a perturbed term with the first-order Casimir operator of the U(5) limit to the SU(3) Hamiltonian in the IBM. We compared the calculation results with the experimental data of $$N=90$$ isotones. Finally, the potential of the Bohr Hamiltonian was represented by a harmonic oscillator; consequently, the structure of $$N=90$$ isotones could be expressed in the closed form by an approximate separation of variables. These nuclear structure prediction results were applied to elucidate the low-lying energy and E2 transitions in isotones.

Electromagnetic transitions between low-lying states in deformed even-even 148–154Sm nuclei were investigated within the framework of the SU(3) limit of the neutron-proton interacting boson model. To explain the M1 transitions between low-lying states and the E2 transition between interband states, we applied first-order perturbation theory by adding the proton-neutron quadrupole interaction Qπ· Qv that breaks F-spin symmetry in the dynamical symmetric SU(3) Hamiltonian as a perturbed term. We calculated the transition probabilities of the M1 and the E2 transition operators between low-lying interband states by using the 1/N expansion methods. The matrix elements of the proton-neutron quadrupole interaction Qπ· Qv were directly calculated, as shown in a previous study, by applying the F-spin operator Qπ· Qa instead of Qπ· Qv. The results of the present study for electromagnetic transitions were applied to some deformed even-even Samarium nuclei, and these theoretical results could effectively describe the experimental data.

An analysis of \({}^{74-82}\mathrm{{Se}}\) within the framework of the interacting boson model (IBM) and the few-body aspects are presented. Selenium isotopes with mass number (A) ranging from 74 to 82 are typical vibrational nuclei that can be explained by the U(5) limit of the IBM. However, they are not fully symmetrical nuclei, and are represented by adding a perturbation term to account for the symmetry breaking. These nuclei tend to exhibit properties of a harmonic oscillator and an asymmetric deformed rotor. The low-energy level and B(E2) ratios are calculated using the IBM via direct diagonalization of the perturbed Hamiltonian and the E(5) symmetry breaking. Comparisons between the theoretical and experimental results of the even-even selenium isotopes with A ranging from 74 to 82 are presented.

The nuclear structure of deformed nuclei has been studied using the interacting boson model (IBM). In this study, energy levels and E2 transition probabilities were determined for even nuclei in the Nd/Sm/Gd/Dy chains which have a transition characteristic between the rotational, SU(3) and vibrational, U(5) limits. The structure of the nuclei exhibits a slight breaking of the SU(3) symmetry in the direction of U(5), and therefore, we add the d-boson number operator n d , which is the main term of the U(5) symmetric Hamiltonian, to the SU(3) Hamiltonian of the IBM. The calculated results for low-lying energy levels and E2 transition rates in Nd/Sm/Gd/Dy isotopes are in reasonably good agreement with known experimental results.

G protein-coupled receptors (GPCRs) are a major membrane receptor family with important physiological and pathological functions. In the classical signaling pathway, ligand-activated GPCRs couple to G proteins, thereby inducing G protein-dependent signaling pathways and phosphorylation by G protein-coupled receptor kinases (GRKs). This leads to an interaction with arrestins, which results in GPCR desensitization. Recently, non-classical GPCR signaling pathways, mediated by GPCR-bound arrestins, have been identified. Consequently, arrestins play important roles in GPCR signaling not only with respect to desensitization but also in relation to G protein-independent signal transduction. These findings have led to efforts to develop functionally biased (i.e. signal transduction biased) GPCR-targeting drugs. One of these efforts is aimed at understanding the structural mechanism of functionally biased GPCR signaling, which includes understanding the G protein-selectivity or arrestin-selectivity of GPCRs. This goal has not yet been achieved; however, great progress has been made during the last 3 years toward understanding the structural mechanism of GPCR-mediated arrestin activation. This review will discuss the recent breakthroughs in the conformational understanding of GPCR-arrestin interaction.

The cell membrane (NCX) and mitochondrial (NCLX) Na

G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.

The abscisic acid (ABA) signaling pathway is regulated by clade A type 2C protein phosphatases (PP2CAs) in plants. In the presence of ABA, PP2Cs release stress/ABA-activated protein kinases by binding to ABA-bound receptors (PYL/RCARs) for activation. Although the wedging tryptophan in PP2Cs is critical in the interaction with PYL/RCARs in Arabidopsis and rice, it remains elusive as to how other interface regions are involved in the interaction. Here, we report the identification of a conserved region on PP2Cs, termed the VxGΦL motif, which modulates the interaction with PYL/RCARs through its second and fourth residues. The effects of the second and fourth residues on the interaction of OsPP2C50 with several OsPYL/RCAR proteins were investigated by systematic mutagenesis. One OsPP2C50 mutant, VFGML ("FM") mutant, lowered the affinity to OsPYL/RCAR3 by ∼15-fold in comparison with the wild-type. Comparison of the crystal structures of wild-type OsPP2C50:ABA:OsPYL/RCAR3 with those composed of FM mutant revealed local conformational changes near the VxGΦL motif, further supported by hydrogen-deuterium exchange mass spectrometry. In rice protoplasts, ABA signaling was altered by mutations in the VxGΦL motif. Transgenic Arabidopsis plants overexpressing OsPP2C50 and OsPP2C50FM showed altered ABA sensitivity. Taken together, the VxGΦL motif of PP2Cs appears to modulate the affinity of PP2Cs with PYL/RCARs and thus likely to alter the ABA signaling, leading to the differential sensitivity to ABA in planta.

An investigation has been made of the behaviour of nuclear structure as a function of an increase in neutron number from 224 Th to 234 Th. Thorium of mass number 234 is a typical rotor nucleus that can be explained by the SU(3) limit of the interacting boson model(IBM) in the algebraic nuclear model. Furthermore, 224−232 Th lie on the path of the symmetry-breaking phase transition. Moreover, the nuclear structure of 224 Th can be explained using X(5) symmetry. However, as 226−230 Th nuclei are not fully symmetrical nuclei, they can be represented by adding a perturbed term to express symmetry breaking. Through the following three calculation steps, we identified the tendency of change in nuclear structure. Firstly, the structure of 232 Th is described using the matrix elements of the Hamiltonian and the electric quadrupole operator between basis states of the SU(3) limit in IBM. Secondly, the low-lying energy levels and E2 transition ratios corresponding to the observable physical values are calculated by adding a perturbed term with the first-order Casimir operator of the U(5) limit to the SU(3) Hamiltonian in IBM. We compared the results with experimental data of 224−234 Th. Lastly, the potential of the Bohr Hamiltonian is represented by a harmonic oscillator, as a result of which the structure of 224−234 Th could be expressed in closed form by an approximate separation of variables. The results of these theoretical predictions clarify nuclear structure changes in Thorium nuclei over mass numbers of practical significance.

Purpose: The study was done to define complementary and alternative medicine (CAM) use among primary brain tumor patients and factors influencing use. Methods: The study was conducted with convenience sampling in 5 neuro-oncology centers in Seoul and Gyeonggi Province. Data were collected using a 25-item questionnaire developed by the researchers. Results: Of 250 patients approached, 231 (92.4%) agreed to participate. Overall, 26.8% of the respondents used CAM and the average cost for CAM use was 300,000 KRW. More than 72% of CAM users did not disclose CAM use to their health care professionals. The most frequently used therapy was natural products including mushrooms. Logistic regression analysis revealed that an education level of university or higher (OR

There are three subtypes of vertebrate inositol 1,4,5-trisphosphate receptor (IP 3 R), a Ca 2+ -release channel on the ER membrane-IP 3 R1, IP 3 R2, and IP 3 R3-each of which has a distinctive role in disease development. To determine the subtype-specific IP 3 -binding mechanism, we compared the thermodynamics, thermal stability, and conformational dynamics between the N-terminal regions of IP 3 R1 (IP 3 R1-NT) and IP 3 R3 (IP 3 R3-NT) by performing circular dichroism (CD), isothermal titration calorimetry (ITC), and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Previously determined crystal structures of IP 3 R1-NT and HDX-MS results from this study revealed that both IP 3 R1 and IP 3 R3 adopt a similar IP 3 -binding mechanism. However, several regions, including the α- and β-interfaces, of IP 3 R1-NT and IP 3 R3-NT show significantly different conformational dynamics upon IP 3 binding, which may explain the different IP 3 -binding affinities between the subtypes. The importance of the interfaces for subtype-specific IP 3 binding is also supported by the different dynamic conformations of the two subtypes in the apo-states. Furthermore, IP 3 R1-NT and IP 3 R3-NT show different IP 3 -binding affinities and thermal stabilities, but share similar thermodynamic properties for IP 3 binding. These results collectively provide new insights into the mechanism underlying IP 3 binding to IP 3 Rs and the subtype-specific regulatory mechanism.

Free fatty acid receptor 4 (FFA4) has been reported to be a receptor for n-3 fatty acids (FAs). Although n-3 FAs are beneficial for bone health, a role of FFA4 in bone metabolism has been rarely investigated. We noted that FFA4 was more abundantly expressed in both mature osteoclasts and osteoblasts than their respective precursors and that it was activated by docosahexaenoic acid. FFA4 knockout (Ffar4(-/-)) and wild-type mice exhibited similar bone masses when fed a normal diet. Because fat-1 transgenic (fat-1(Tg+)) mice endogenously converting n-6 to n-3 FAs contain high n-3 FA levels, we crossed Ffar4(-/-) and fat-1(Tg+) mice over two generations to generate four genotypes of mice littermates: Ffar4(+/+);fat-1(Tg-), Ffar4(+/+);fat-1(Tg+), Ffar4(-/-);fat-1(Tg-), and Ffar4(-/-);fat-1(Tg+). Female and male littermates were included in ovariectomy- and high-fat diet-induced bone loss models, respectively. Female fat-1(Tg+) mice decreased bone loss after ovariectomy both by promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption than their wild-type littermates, only when they had the Ffar4(+/+) background, but not the Ffar4(-/-) background. In a high-fat diet-fed model, male fat-1(Tg+) mice had higher bone mass resulting from stimulated bone formation and reduced bone resorption than their wild-type littermates, only when they had the Ffar4(+/+) background, but not the Ffar4(-/-) background. In vitro studies supported the role of FFA4 as n-3 FA receptor in bone metabolism. In conclusion, FFA4 is a dual-acting factor that increases osteoblastic bone formation and decreases osteoclastic bone resorption, suggesting that it may be an ideal target for modulating metabolic bone diseases.

G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein–protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.

Renal ischemic events open tight junctions and disrupt epithelial polarity. The purpose of this study was to examine the effects of ischemia-reperfusion (IR) injury on expression and distribution of the tight junction proteins, occludin and ZO-1, in the rat kidney. IR injury was induced by clamping both renal pedicles for 30 min and animals were killed at 6 h after the reperfusion. IR injury decreased blood bicarbonate level, but did not persistently alter pH, Na(+), K(+), or Cl(-). In control kidneys, occludin immunoreactivity was intense in the tight junctions in the thick ascending limb, distal convoluted tubule, and collecting duct, moderate in the thin limbs of the loop of Henle, and was not detected in the proximal tubule, glomerulus, and blood vessels. ZO-1 was expressed in the same sites in which occludin was expressed, and additionally was also expressed in the proximal tubule, glomerulus, and vascular endothelial cells. IR kidneys exhibited damaged renal tubular epithelial cells in both proximal tubule and collecting duct segments in the outer medulla. In the collecting duct, the response of intercalated cells and principal cells differed. Following IR injury, intercalated cells, but not principal cells, lost their normal epithelial polarity and were frequently extruded into the tubule lumen. Occludin, instead of being localized to tight junctions, was localized diffusely in the cytoplasm in intercalated cells of IR kidneys. Principal cells, in contrast, were not detectably affected and neither occludin nor ZO-1 expression were altered in response to IR injury. The normal localization of ZO-1 expression to tight junction sites in both the proximal tubule and collecting duct was altered in response to IR, and, instead, ZO-1 expression was present diffusely in the cytoplasm. IR injury did not alter detectably either occludin or ZO-1 localization to the tight junction of the thick ascending limb cells. The abundance of total occludin protein by immunoblot analysis was not changed with IR injury. These results demonstrate that renal IR injury causes tight junction disruptions in both the proximal tubule and the collecting duct, and that altered distribution of the tight junction protein, occludin, may play a critical role in the collecting duct dysfunction which IR induces.

The aim of this study was to know whether leaf nitrate can be a substitute of total leaf N to justify plant N status and how nitrate influences macro elements uptake and physiological parameters of tomato plants under different nitrogen levels. Leaf nitrate content decreased in low N, while showed similar value with the control in high N, ranging from 55 to 70 mg g -1 . Differences in nitrate supply led to nitrate-dependent increases in macro elements, particularly cations, while gradual decrease in P. Physiological parameters, photosynthesis rates and antioxidants, greatly responded in N deficient conditions rather than high N, which didn’t show any significant differences compared the control. Considering nitrogen forms and physiological parameters, total-N in tomato plants represented positive relation with growth (shoot dry weight), nitrate and CO 2 assimilation, whereas negative relation with lipid peroxidation.

Ammonia metabolism is a primary component of acid-base homeostasis but is incompletely developed at time of birth. Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg) are recently recognized ammonia transporter family members expressed in the mammalian kidney. This study's purpose was to establish the expression and localization of Rhbg and Rhcg during kidney development. We examined kidneys from fetal days 16 ( E16), 18 ( E18), and 20 ( E20), and from the first 21 days of postnatal development. Rhbg was expressed initially at E18, with expression only in the connecting tubule (CNT); at E20, Rhbg was expressed in both the CNT and the medullary collecting duct (MCD). In contrast, Rhcg was first expressed at E16 with basal expression in the ureteric bud; at E18, it was expressed in a subset of CNT cells with an apical pattern, followed by apical and basolateral expression in the MCD at E20. In the cortex, Rhbg and Rhcg expression increased in the CNT before expression in the cortical collecting duct during fetal development. In the MCD, both Rhbg and Rhcg expression was initially in cells in the papillary tip, with gradual removal from the tip during the late fetal period and transition during the early neonatal period to an adult pattern with predominant expression in the outer MCD and only rare expression in cells in the initial inner MCD. Double-labeling with intercalated cell-specific markers identified that Rhbg and Rhcg were expressed initially in CNT cells, CNT A-type intercalated cells and non-A, non-B intercalated cells, and in MCD A-type intercalated cells. We conclude that expression of Rhbg and Rhcg parallels intercalated cell development and that immature Rhbg and Rhcg expression at birth contributes to incomplete ammonia excretion capacity.

The purpose of this study is to investigate the students' preference and the sensibility to fetish fabrics such as leather, fur, velvet, suade, and corduroy, and to analyze differences according to gender of the university students. A questionnaire survey was conducted on 324 university students (male students 52.8%, female students 47.2%) in the Cheongju area, Korea. Frequency, t-test, F-test and factor analysis were used for data analysis. The results were as follows: 1) the subjects preferred velvet, leather and suede fabrics, while they did not prefer corduroy and fur fabrics. 2) Their feelings to fetish fabrics were respectively different depending on the kind of fabrics. 3) There were partially significant differences between the gender in the preference and the sensibility to fetish fabrics. 4) They evaluated that velvet fabrics could express a variety of sensibilities. 5) They considered that fabrics as velvet, leather, and fur have more noble and fanciful images than corduroy.

Granulocytic sarcoma (GS) is defined as a localized tumor mass composed of myeloid blasts and/or immature myeloid cells in an extramedullary site. Usually, GS occurs concomitantly with or after acute myelogenous leukemia (AML), myeloproliferative disorder, or myelodysplastic syndrome. In rare cases, it occurs as a "preleukemic" condition and may precede the onset of AML, which occurs within several months if the patient is not treated with AML-type systemic chemotherapy. Recently, we discovered one case of nonleukemic GS in the small intestine incidentally when intussusception was suspected. The patient visited the emergency department, in October 2006, with symptoms of small-bowel obstruction. Intussusception due to a small-intestinal mass was suspected after evaluation, and small-intestine segmental resection was performed. The patient had no previous history of leukemia, and immunohistochemical staining was used to diagnose GS. Bone-marrow biopsy performed subsequently revealed no lesions that could be suspected as leukemia. The patient received three cycles of chemotherapy, applied as for AML (cytosine arabinoside and anthracycline), and is currently, as of October 29, 2007, showing no other marked indisposition; he has been disease-free for 12 months.