In the present study, expression and localization of PLD2 were examined in mouse submandibular glands in situ in comparison with those of PLD1 previously reported by the present authors. In immunoblots, PLD2-expression was high at postnatal 0 week (P0W) and P2W, and at P4W decreased considerably with the decrease of the male gland more marked, and at P8W it was undetectable in the male gland, but remained faint in the female. In the male gland at P8W after castration at P6W, PLD2-expression reappeared faintly. In the female glands with daily injections of testosterone started at P6W, at P8W the expression was undetectable. In immunohistochemistry, PLD2 was localized throughout immature duct epithelia without distinct regional differences in its intensity at P0W and P2W. PLD2-localization was weak and diffuse in granular convoluted tubules at P4W and negligible there at P8W in the female gland, while it was at negligible levels in the tubules at P4W and at undetectable levels there at P8W in the male gland. The expression changes detected in immunoblots after castration or testosterone injection were duplicated in granular convoluted tubules in immunohistochemistry. The present findings suggest that PLD2 is dominantly involved in proliferation/differentiation/apoptosis of most immature ductal cells at early postnatal and at least perinatal stages, different from PLD1. This also indicates that the female-dominant sexual dimorphism of PLD2-expression occurs in synchrony of differentiation of granular convoluted tubules at puberty and at young adulthood, similar to PLD1 though at much lower levels.

T315I mutation found in chronic myelogenous leukemia (CML) and Ph + ALL patients is the most serious one among resistance against BCR/ABL kinase inhibitors including imatinib and is only responsive to ponatinib (PNT). However, the novel strategy is required to reduce life-threatening adverse effects of PNT including ischemic cardiovascular disease. We examined the mechanism of PNT-induced cytotoxicity against a T315I(+) Ph + ALL cell line, TccY/Sr. PNT induced apoptosis (increased sub G1 cells, and cleaved caspase3 and PARP), and suppressed protein expression of MCL1, cyclin D2 and c-myc, which were reversed by a proteasome inhibitor, MG132, suggesting enhanced proteasomal degradation by PNT. Among BCL2 family inhibitors, MCL1 inhibitors (maritoclax and AZD5991) robustly induced cell death, showing the MCL1-dependent survival of TccY/Sr cells. Decreased MCL1 and c-myc expression by PNT was also observed in T315I(+) MEGA2/STIR cells. PNT suppressed PI3K activation followed by AKT inhibition and GSK3 dephosphorylation. PI3K/AKT inhibitors mimicked PNT, suggesting that PI3K/AKT signaling is important for survival of TccY/Sr cells. Moreover, GSK3 inhibitor (SB216763) reduced PNT-induced cytotoxicity and degradation of c-myc and MCL1. AZD5991 exhibited the synergistic action with PNT, anti-cancer drugs and venetoclax (BCL2 inhibitor), suggesting the utility of MCL1 inhibitor alone or in combination as a future clinical option for Ph + leukemia patients.

Background We compared the dosimetric and clinical outcomes of VMAT and 3D-CRT in breast cancer patients undergoing regional nodal irradiation (RNI) to determine the effectiveness of VMAT. Methods This retrospective cohort study included breast cancer patients who underwent adjuvant radiotherapy with RNI between July 2016 and September 2022. Patients were assigned to receive either 3D-CRT or VMAT based on the laterality of their cancer. Right-sided breast cancer received 3D-CRT, while left-sided breast cancer received VMAT. Radiotherapy consisted of a dose of 50 Gy/25 fr to the chest wall or breast and the regional nodes. Dosimetric parameters, adverse events, and survival were compared between 3D-CRT and VMAT. Results A total of 126 patients were included in the analysis, with 64 in the 3D-CRT group and 62 in the VMAT group. VMAT showed better coverage of the clinical target volume (P < 0.01). Among patients who received internal mammary node irradiation, VMAT resulted in a lower total lung V20 Gy compared to 3D-CRT (17% vs. 11%, P < 0.01), whereas total lung V5 Gy was higher for VMAT (27% vs. 34%, P < 0.01). The incidence of grade 2 acute dermatitis was lower in the VMAT group (27%) than in the 3D-CRT group (47%) (P= 0.02). The 5-year overall survival and breast cancer-specific survival rates were 87% and 90% in the 3D-CRT group and 100% and 100% in the VMAT group, respectively (P = 0.06 and 0.09). Conclusions VMAT showed better target coverage and less acute radiation dermatitis compared to 3D-CRT.

To clarify the signal transduction mechanism in the differentiation and secretion of salivary glandular cells, the present study was attempted to examine in the submandibular gland (SMG) of mice, the expression and localization of phospholipase D1 (PLD1), one of the important effector molecules working in response to the activation of intramembranous receptors by first messengers. In immunoblotting analysis, the expression of PLD1 was high at postnatal 4 weeks (P4W) and decreased at P8W, and it was at negligible levels at newborn stage (P0W) and postnatal 2 weeks (P2W). The expression of PLD1 was greater in females, and it was suppressed by administration of testosterone to female mice. In immuno-light microscopy, immunoreactivity for PLD1 at P4W was moderate to intense, in the forms of dots and globules mainly in the apical domains of immature granular convoluted tubule (GCT)-cells localized largely in the proximal portion of the female GCT. By P8W, it decreased in intensity and remained weak to moderate along the apical plasmalemma of cells throughout the course of the female GCT, whereas it was faint throughout the GCT of the male SMG at P4W and negligible at P8W. In immuno-electron microscopy, immature GCT-cells characterized by electron-lucent granules were immunoreactive and the immunoreactive materials were deposited close to, but not within, those granules. Typical GCT cells, characterized by electron-dense granules, were immunonegative. No significant immunoreaction for PLD1 was seen in acini of SMGs of either sex at any time point examined. It is suggested that PLD1 is involved in the signaling for secretion of immature GCT cells and influences differentiation of these cells, probably through their own secretory substances.

Resveratrol (RSV) has recently attracted keen interest because of its pleiotropic effects. It exerts a wide range of health-promoting effects. In addition to health-promoting effects, RSV possesses anti-carcinogenic activity. However, a non-physiological concentration is needed to achieve an anti-cancer effect, and its in vivo bioavailability is low. Therefore, the clinical application of phytochemicals requires alternative candidates that induce the desired effects at a lower concentration and with increased bioavailability. We previously reported a low IC50 of vaticanol C (VTC), an RSV tetramer, among 12 RSV derivatives (Ito T. et al, 2003). However, the precise mechanism involved remains to be determined. Here, we screened an in-house chemical library bearing RSV building blocks ranging from dimers to octamers for cytotoxic effects in several leukemia and cancer cell lines and their anti-cancer drug-resistant sublines. Among the compounds, VTC exhibited the highest cytotoxicity, which was partially inhibited by a caspase 3 inhibitor, Z-VAD-FMK. VTC decreased the expression of sphingosine kinase 1, sphingosine kinase 2 and glucosylceramide synthase by transcriptional or post-transcriptional mechanisms, and increased cellular ceramides/dihydroceramides and decreased sphingosine 1-phosphate (S1P). VTC-induced sphingolipid rheostat modulation (the ratio of ceramide/S1P) is thought to be involved in cellular apoptosis. Indeed, exogenous S1P addition modulated VTC cytotoxicity significantly. A combination of SPHK1, SPHK2, and GCS chemical inhibitors induced sphingolipid rheostat modulation, cell growth suppression, and cytotoxicity similar to that of VTC. These results suggest the involvement of sphingolipid metabolism in VTC-induced cytotoxicity, and indicate VTC is a promising prototype for translational research.

Abstract Background Early diastolic suction is an important determinant of early diastolic function. Previous studies using color Doppler M-mode and speckle-tracking echocardiography have shown left ventricular (LV) early diastolic LV untwisting rate is directly related to LV intra-ventricular pressure gradient (IVPG) between LV base and apex during early diastole. Recent introduction of vector flow mapping (VFM) using combination of color Doppler and speckle-tracking echocardiography provides noninvasive and feasible assessment of early diastolic IVPG shortly after aortic valve closure including isovolmic relaxation period (ED-IVPG) in humans. However, relationship between VFM–derived ED-IVPG and early diastolic LV untwisting rate has not been well investigated. Purpose The purpose of this study was to examine relationship between ED-IVPG estimated by VFM and LV untwisting rate by speckle-tracking echocardiography. Methods The study population consists of 66 patients without segmental wall motion abnormality, significant valvular diseases, and atrial fibrillation who underwent echocardiography for evaluation of LV function (age: 60±15 years, LVEF: 49±16%). From the apical long-axis views by color Doppler echocardiography, we analyzed peak ED-IVPG between LV base and apex just after aortic valve closure (figure) using commercially available VFM analysis software (DAS-RS1, Hitachi). We assessed peak early diastolic LV untwisting rate and LV torsion from LV basal and apical short-axis view by speckle-tracking echocardiography. We evaluated correlation between ED-IVPG and LV untwisting rate. We also evaluated correlation between ED-IVPG and peak systolic LV torsion, LV end-diastolic (EDV) and end-systolic volumes (ESV), ejection fraction (EF), early diastolic velocity (E) of LV inflow, average early diastolic velocity (e') of mitral annulus, and average E/e'. Results In all the study patients, ED-IVPG was successfully and quickly evaluated. 1) ED-IVPG correlated well with peak LV untwisting rate (r=0.64, p<0.0001). 2) ED-IVPG significantly correlated with LV torsion, LVEDV, LVESV, and LVEF (r=0.47, r=−0.48, r=−0.46, and r=0.48, respectively, p<0.001). 3) There were no significant correlations between ED-IVPG and other indexes including E, average e', and average E/e'. According to receiver operating characteristic analysis, the best cut-off value of ED-IVPG for determining impaired LV untwisting rate (<80 degrees/s) was found at 0.42 mmHg (sensitivity 81%, specificity 76%, and area under the curve 0.86) ED-IVPG measurement by VFM Conclusions The present results showed that noninvasive VFM-derived peak ED-IVPG shortly after aortic valve closure is related to early diastolic peak LV untwisting rate. ED-IVPG easily and quickly estimated by VFM may be used as an additional index for LV diastolic function.

Background: Previous studies have shown that there are left ventricular (LV) intra-ventricular pressure gradients (IVPG) between LV apex and outflow tract during early systole, and between LV base and apex during early diastole, using invasive catheter method and noninvasive color M-mode echocardiography.These IVPGs have been reported to be affected by LV systolic and diastolic function.Recent introduction of vector flow mapping (VFM) using combination of color Doppler and tissue Doppler echocardiography provides noninvasive and feasible assessment of LV IVPG in the clinical setting.However, relationship between IVPG estimated by VFM and LV systolic and diastolic function has not been well evaluated.Purpose: The purpose of this study was to examine the relationship between LV IVPG during early systole and early diastole estimated by VFM and LV systolic and diastolic function. Methods:The study population consists of 53 subjects without myocardial infarction, significant valvular diseases, and arrhythmia (age: 67±11 years, LVEF: 32±8%).Apical long-axis views were recorded by color Doppler echocardiography (Prosound F 75 and α10, Hitachi.Peak IVPG between apex and LV outflow tract during early systole after closing mitral valve closure (ES-IVPG, figure A) and peak IVPG between mitral annulus and apex during early diastole after mitral valve opening (ED-IVPG, figure B) were quickly assessed using commercially available VFM analysis software (DAS-RS1, Hitachi).Correlations between each IVPG and LV systolic functional index [end-diastolic (EDV) and end-systolic volume (ESV)] were examined.We also evaluated correlation between ED-IVPG and diastolic functional index (E of LV inflow velocity, e' of mitral annular velocity, and E/e').Results: 1) Peak ES-IVPG significantly correlated with LVEDV (r=-0.40,p=0.0044),LVESV (r=-0.44,p=0.0014) and LVEF (r=-0.41,p=0.0023).2) Peak ED-IVPG significantly correlated with LVEDV (r=-0.35,p=0.0139),LVESV (r=-0.47,p=0.0007) and LVEF (r=-0.52,p<0.001).3) Peal ED-IVPG significantly correlated with E (r=0.50, p=0.0002), e' (r=0.77,p<0.0001) and E/e' (r=-0.48,p=0.0005).Peak ES-and ED-IVPG measurement by VFM Conclusions: The present study showed 1) both early systolic and early diastolic peak LV IVPG by VFM analysis were affected by LV systolic function, and 2) early diastolic peak LV IVPG by VFM was strongly affected by early diastolic mitral annular velocity suggesting myocardial relaxation.LV IVPG estimated by VFM could be used as an additional new index for LV systolic and diastolic function in the clinical setting.

Imatinib (IMT), a specific tyrosine kinase inhibitor (TKI), has drastically changed the treatment strategy for Ph+ ALL (Philadelphia chromosome-positive acute lymphoblastic leukemia). However, TKI resistance remains a serious problem for patient prognosis. Here, a Ph+ ALL cell line NphA2 and the IMT-resistant subline NphA2/STIR were analyzed to identify a potential novel treatment strategy. We also examined other Ph+ ALL cells, MR87 and its IMT-resistant subline, MR87/STIR. IMT induced apoptosis of NphA2 and MR87 but had no effect on resistant sublines. Increased phosphorylated ERK and BCL2, but not BCL-XL, were observed in NphA2/STIR compared with NphA2. NphA2/STIR but not NphA2 was moderately sensitive to U0126, an ERK inhibitor. Interestingly, SP600125, a JNK inhibitor, was potent in cell growth inhibition and apoptosis induction of both parental and IMT-resistant NphA2 and MR87 cells. Moreover, NphA2 and MR87 and their IMT-resistant sublines were sensitive to ABT-199, a specific BCL2 inhibitor. The combination of SP600125 and ABT-199 synergistically suppressed both parental and IMT-resistant cells, including one with T315I mutation, suggesting that Ph+ ALL exhibits high sensitivity to ABT-199 and SP600125 regardless of TKI resistance. This combination might be a possible therapeutic strategy for Ph+ ALL in the future.

Paclitaxel (PTX) is a microtubule-targeting drug widely used for the treatment of a variety of cancers. However, drug resistance can emerge after a series of treatments, and this can seriously affect the patient's prognosis. Here, we analyzed the mechanism of PTX resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. Compared with PC3, PC3-PR exhibited some unique phenotypes that might be associated with PTX resistance, including decreased expression of acetylated α-tubulin and the cell cycle regulator p21, and increased expression of βIII tubulin, histone deacetylase 6 (HDAC6), and the anti-apoptotic protein Bcl2. The drug exporters MDR1 and MRP1 were not involved in PTX resistance. Although cabazitaxel (CTX), a novel taxoid, has been reported to overcome PTX resistance, its mechanism of action is unknown. We found that treatment of PC3-PR cells with CTX induced expression of acetylated α-tubulin and p21, but not the related regulators p27, p15, and p16 or the Bcl2 family proteins. The pan-HDAC inhibitors trichostatin A and suberanilohydroxamic acid and the HDAC6-specific inhibitor tubacin inhibited PC3-PR proliferation and increased expression of p21 and acetylated α-tubulin in a manner similar to CTX. Our data shed light on the cellular response to PTX and CTX.

Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and SIP by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5'-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. (C) 2016 Elsevier Inc. All rights reserved.

Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression.

Compiled abundances of juvenile corals revealed no change over time in the Pacific, but a decline in the Caribbean. Using these analyses as a rationale, we explored recruitment and post-settlement success in determining coral cover using studies in the Caribbean (St John, Bonaire) and Pacific (Moorea, Okinawa). Juvenile corals, coral recruits, and coral cover have been censused in these locations for years, and the ratio of juvenile (J) to recruiting (R) corals was used to measure post-settlement success. In St John and Bonaire, coral cover was stable but different between studies, with the ratio of the density of juveniles to density of recruits (J:R) ~0.10; in Moorea, declines in coral cover were followed by recovery that was related to the density of juvenile corals 3 years before, with J:R ~0.40; and in Okinawa, a decline in coral cover in 1998 was followed by a slow recovery with J/R ~0.01. Coral cover was associated positively with juvenile corals in St John, and in Okinawa, the density of juvenile corals was associated positively with recruits the year before. J:R varied among studies, and standardised densities of juvenile corals declined in the Caribbean, but increased in the Pacific. These results suggest that differences in the post-settlement success may drive variation in coral community structure.

An artificial diet mixed with persimmon wine residue (PR) was used in cultivating ayu fish in an effort to improve its taste and body color, and for the effective use of leftover PR. There were no significant differences in the fatty acid composition of dorsal meat between PR-feeding and control groups. Measurement of color differences in yellow spots, dorsal fins, ventral fins, and caudal fins revealed that body color of the PR-feeding group was significantly improved compared to the control group. A survey was administered regarding ayu overall, and the survey items "appearance" and "taste" were rated significantly higher in the PR-feeding group than in the control group. From the above mentioned results, it is suggested that as an effective use for PR, a PR-mixed feed can improve preferential attributes of ayu such as body color and taste.

Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5' SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture.

Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between -49bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.

The pentaquark Theta(+) has been searched for via the pi(-) p --> K- X reaction with beam momenta of 1.92 and 2.01 GeV/c at J-PARC. A missing mass resolution of 2 MeV (FWHM) was achieved but no sharp peak structure was observed. The upper limits on the production cross section averaged over the scattering angle from 2 degrees to 15 degrees in the laboratory frame were found to be less than 0.28 mu b/sr at the 90% confidence level for both the 1.92- and 2.01- GeV/c data. The systematic uncertainty of the upper limits was controlled within 10%. Constraints on the Theta(+) decay width were also evaluated with a theoretical calculation using an effective Lagrangian. The present result implies that the width should be less than 0.36 and 1.9 MeV for the spin-parity of 1/2(+) and 1/2(-), respectively.