Temporal involvement of phosphatidylinositol 4-phosphate 5-kinase γ in differentiation of Z-bands and myofilament bundles as well as intercalated discs in mouse heart at mid-gestation.

Considering the occurrence of serious heart failure in a gene knockout mouse of PIP5Kγ and in congenital abnormal cases in humans in which the gene was defective as reported by others, the present study attempted to localize PIP5Kγ in the heart during prenatal stages. It was done on the basis of the supposition that phenotypes caused by gene mutation of a given molecule are owed to the functional deterioration of selective cellular sites normally expressing it at significantly higher levels in wild mice. PIP5Kγ-immunoreactivity was the highest in the heart at E10 in contrast to almost non-significant levels of the immunoreactivity in surrounding organs and tissues such as liver. The immunoreactivity gradually weakened in the heart with the prenatal age, and it was at non-significant levels at newborn and postnatal stages. Six patterns in localization of distinct immunoreactivity for PIP5Kγ were recognized in cardiomyocytes: (1) its localization on the plasma membranes and subjacent cytoplasm without association with short myofibrils and (2) its localization on them as well as short myofibrils in association with them in cardiomyocytes of early differentiation at E10; (3) its spot-like localization along long myofibrils in cardiomyocytes of advanced differentiation at E10; (4) rare occurrences of such spot-like localization along long myofibrils in cardiomyocytes of advanced differentiation at E14; (5) its localization at Z-bands of long myofibrils; and (6) its localization at intercellular junctions including the intercalated discs in cardiomyocytes of advanced differentiation at E10 and E14, especially dominant at the latter stage. No distinct localization of PIP5Kγ-immunoreactivity of any patterns was seen in the heart at E18 and P1D. The present finding suggests that sites of PIP5Kγ-appearance and probably of its high activity in cardiomyocytes are shifted from the plasma membranes through short myofibrils subjacent to the plasma membranes and long myofibrils, to Z-bands as well as to the intercalated discs during the mid-term gestation. It is further suggested that PIP5Kγ is involved in the differentiation of myofibrils as well as intercellular junctions including the intercalated discs at later stages of the mid-term gestation. Failures in its involvement in the differentiation of these structural components are thus likely to cause the mid-term gestation lethality of the mutant mice for PIP5Kγ.

Super-strong magnetic field-dominated ion beam dynamics in focusing plasma devices.

High energy density physics is the field of physics dedicated to the study of matter and plasmas in extreme conditions of temperature, densities and pressures. It encompasses multiple disciplines such as material science, planetary science, laboratory and astrophysical plasma science. For the latter, high energy density states can be accompanied by extreme radiation environments and super-strong magnetic fields. The creation of high energy density states in the laboratory consists in concentrating/depositing large amounts of energy in a reduced mass, typically solid material sample or dense plasma, over a time shorter than the typical timescales of heat conduction and hydrodynamic expansion. Laser-generated, high current-density ion beams constitute an important tool for the creation of high energy density states in the laboratory. Focusing plasma devices, such as cone-targets are necessary in order to focus and direct these intense beams towards the heating sample or dense plasma, while protecting the proton generation foil from the harsh environments typical of an integrated high-power laser experiment. A full understanding of the ion beam dynamics in focusing devices is therefore necessary in order to properly design and interpret the numerous experiments in the field. In this work, we report a detailed investigation of large-scale, kilojoule-class laser-generated ion beam dynamics in focusing devices and we demonstrate that high-brilliance ion beams compress magnetic fields to amplitudes exceeding tens of kilo-Tesla, which in turn play a dominant role in the focusing process, resulting either in a worsening or enhancement of focusing capabilities depending on the target geometry.

Blockade of the KATP channel Kir6.2 by memantine represents a novel mechanism relevant to Alzheimer's disease therapy.

Here, we report a novel target of the drug memantine, ATP-sensitive K+ (KATP) channels, potentially relevant to memory improvement. We confirmed that memantine antagonizes memory impairment in Alzheimer's model APP23 mice. Memantine increased CaMKII activity in the APP23 mouse hippocampus, and memantine-induced enhancement of hippocampal long-term potentiation (LTP) and CaMKII activity was totally abolished by treatment with pinacidil, a specific opener of KATP channels. Memantine also inhibited Kir6.1 and Kir6.2 KATP channels and elevated intracellular Ca2+ concentrations in neuro2A cells overexpressing Kir6.1 or Kir6.2. Kir6.2 was preferentially expressed at postsynaptic regions of hippocampal neurons, whereas Kir6.1 was predominant in dendrites and cell bodies of pyramidal neurons. Finally, we confirmed that Kir6.2 mutant mice exhibit severe memory deficits and impaired hippocampal LTP, impairments that cannot be rescued by memantine administration. Altogether, our studies show that memantine modulates Kir6.2 activity, and that the Kir6.2 channel is a novel target for therapeutics to improve memory impairment in Alzheimer disease patients.

Boosting laser-ion acceleration with multi-picosecond pulses.

Using one of the world most powerful laser facility, we demonstrate for the first time that high-contrast multi-picosecond pulses are advantageous for proton acceleration. By extending the pulse duration from 1.5 to 6 ps with fixed laser intensity of 1018 W cm-2, the maximum proton energy is improved more than twice (from 13 to 33 MeV). At the same time, laser-energy conversion efficiency into the MeV protons is enhanced with an order of magnitude, achieving 5% for protons above 6 MeV with the 6 ps pulse duration. The proton energies observed are discussed using a plasma expansion model newly developed that takes the electron temperature evolution beyond the ponderomotive energy in the over picoseconds interaction into account. The present results are quite encouraging for realizing ion-driven fast ignition and novel ion beamlines.

Slowdown mechanisms of ultraintense laser propagation in critical density plasma.

We use one- and two-dimensional particle-in-cell simulations to demonstrate that the propagation of an ultraintense laser (I=10(19)W/cm(2)) in critical density plasma can be interfered with by a high density plasma wall region generated at the propagation front. When the electron flow speed of the wall region exceeds a certain relativistic threshold, the region behaves as an overdense plasma due to a decrease of the effective critical density. The region forms then very small overdense plasma islands. The islands impede the propagation intermittently and slow down the propagation speed significantly.

An electron/ion spectrometer with the ability of low energy electron measurement for fast ignition experiments.

An electron energy spectrometer (ESM) is one of the most fundamental diagnostics in the fast ignition experiment. It is necessary to observe the spectra down to a low energy range in order to obtain the accurate deposition efficiency toward the core. Here, we realize the suitable ESM by using a ferrite magnet with a moderate magnetic field of 0.3 T and a rectangular magnetic circuit covered with a steel plate in the inlet side.

Reduction of both beta cell death and alpha cell proliferation by dipeptidyl peptidase-4 inhibition in a streptozotocin-induced model of diabetes in mice.

AIMS/HYPOTHESIS: Incretins stimulate insulin secretion in a glucose-dependent manner but also promote pancreatic beta cell protection. Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new glucose-lowering treatment that blocks incretin degradation by DPP-4. We assessed whether DPP-4 inhibition suppresses the progression to hyperglycaemia in a low-dose streptozotocin (STZ)-induced diabetic mouse model, and then investigated how DPP-4 inhibition affects islet function and morphology. METHODS: The DPP-4 inhibitor, des-fluoro-sitagliptin (SITA), was administered to mice during and after STZ injections, and in some mice also before STZ. RESULTS: In control mice, STZ resulted in hyperglycaemia associated with impaired insulin secretion and excess glucagon secretion. In SITA-treated STZ mice, these metabolic abnormalities were improved, particularly when SITA administration was initiated before STZ injections. We observed beta cell loss and dramatic alpha cell expansion associated with decreased insulin content and increased glucagon content after STZ administration. In SITA-treated mice, islet architecture and insulin content were preserved, and no significant increase in glucagon content was observed. After STZ exposure, beta cell apoptosis increased before hyperglycaemia, and SITA treatment reduced the number of apoptotic beta cells. Interestingly, alpha cell proliferation was observed in non-treated mice after STZ injection, but the proliferation was not observed in SITA-treated mice. CONCLUSIONS/INTERPRETATION: Our results suggest that the ability of DPP-4 inhibition to suppress the progression to STZ-induced hyperglycaemia involves both alleviation of beta cell death and alpha cell proliferation.

Negative regulation of NaF-induced apoptosis by Bad-CAII complex.

Fluoride is used to prevent caries in dentistry. However, its mechanism of cytotoxicity induction is unclear. This study was undertaken to determine whether sodium fluoride (NaF) induces apoptosis in human oral cells and if so, whether Bad protein is involved in the process. NaF showed higher cytotoxicity and apoptosis-inducing activity against human oral squamous cell carcinoma cells (HSC-2) than against human gingival fibroblasts (HGF). Western blot analysis showed that NaF enhanced the expression and dephosphorylation of Bad protein. This study demonstrates for the first time that Bad protein forms a complex with carbonic anhydrase II (CAII), and NaF stimulates the detachment of CAII from the Bad-CAII complex and the replacement by the formation of Bad-Bcl-2 complex. Knockdown of Bad and CAII mRNA by siRNA inhibited and enhanced the NaF-induced caspase activation, respectively. The present study suggests that CAII negatively regulates the NaF-induced apoptosis by forming a complex with Bad.

Sodium 5,6-benzylidene-L-ascorbate induces oxidative stress, autophagy, and growth arrest in human colon cancer HT-29 cells.

Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT-29 cells with SAA and SBA, and found a significant exposure time-dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC(50) = 5 mM) than SBA (IC(50) = 10 mM). A short-term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase-dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21(+/+) and p21(-/-)), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co-incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N-acetyl-L-cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3-II were found in SBA-treated HT-29 cells; implicating that SBA induced AKT phosphorylation-autophagy and p21-growth arrest in colon cancer HT-29 cells through an NAC-inhibitable oxidative stress pathway.

Fluorescent labeling of both GABAergic and glycinergic neurons in vesicular GABA transporter (VGAT)-venus transgenic mouse.

Inhibitory neurons play important roles in a number of brain functions. They are composed of GABAergic neurons and glycinergic neurons, and vesicular GABA transporter (VGAT) is specifically expressed in these neurons. Since the inhibitory neurons are scattered around in the CNS, it is difficult to identify these cells in living brain preparations. The glutamate decarboxylase (GAD) 67-GFP knock-in mouse has been widely used for the identification of GABAergic neurons, but their GAD67 expression was decreased compared to the wild-type mice. To overcome such a problem and to highlight the function and morphology of inhibitory neurons, we generated four lines of VGAT-Venus transgenic mice (lines #04, #29, #39 and #49) expressing Venus fluorescent protein under the control of mouse VGAT promoter. We found higher expression level of Venus transcripts and proteins as well as brighter fluorescent signal in line #39 mouse brains, compared to brains of other lines examined. By Western blots and spectrofluorometric measurements of forebrain, the line #39 mouse showed stronger GFP immunoreactivity and brighter fluorescent intensity than the GAD67-GFP knock-in mouse. In addition, Venus was present not only in somata, but also in neurites in the line #39 mouse by histological studies. In situ hybridization analysis showed that the expression pattern of Venus in the line #39 mouse was similar to that of endogenous VGAT. Double immunostaining analysis in line #39 mouse showed that Venus-expressing cells are primarily immunoreactive for GABA in cerebral cortex, hippocampus and cerebellar cortex and for GABA or glycine in dorsal cochlear nucleus. These results demonstrate that the VGAT-Venus line #39 mouse should be useful for studies on function and morphology of inhibitory neurons in the CNS.

Partial functional overlap of the three ras genes in mouse embryonic development.

In mammals, three ras genes, H-ras, N-ras and K-ras, encode homologous but distinct 21-kDa Ras proteins. We examined the in vivo functional relationship of the three ras genes in mouse embryonic development by investigating the phenotypes of mice deficient in one or multiple ras genes. H-ras-/- mice and N-ras-/- mice as well as a substantial proportion of H-ras-/-/N-ras-/- mice expressing only the K-ras gene were viable, while K-ras-/- mice were embryonically lethal, as have been reported previously. N-ras-/-/K-ras+/- mice died neonatally, while H-ras-/-/K-ras-/- embryos died much earlier than K-ras homozygous mutant fetuses. To further investigate the functional relationship of the ras genes in embryonic development, we introduced a human H-ras transgene into single or multiple ras mutant mice and found that the transgene rescued mice, including triple ras mutants, from embryonic lethality in association with correction of thin ventricular walls of the heart in null K-ras mutant mice. In situ hybridization revealed that the expression of the H-ras transgene on embryonic day E13.5 and E15.5 was more intense in major organs, including the heart, than those of endogenous ras genes. We therefore conclude that the functions of the ras genes are partially overlapping in mouse embryonic development.

Possible link between glycolysis and apoptosis induced by sodium fluoride.

Fluoride has been used to prevent caries in the dentition, but the possible underlying mechanisms of cytotoxicity induction by this compound are still unclear. Since fluoride is known as an inhibitor of glycolytic enzymes, we investigated the possible connection between NaF-induced apoptosis and glycolysis in human promyelocytic leukemia HL-60 cells. NaF-induced apoptotic cell death is characterized by caspase activation, internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, and production of apoptotic bodies. Higher activation of caspases-3 and -9, as compared with that of caspase-8, suggested the involvement of an extrinsic pathway. Utilization of glucose was nearly halted by NaF, whereas that of glutamine was rather enhanced. NaF enhanced the expression of Bad protein, but not that of Bcl-2 and Bax proteins, and reduced HIF-1alpha mRNA expression. Analysis of these data suggests a possible link between glycolysis and apoptosis.

Interaction between 2-ethoxybenzoic acid (EBA) and eugenol, and related changes in cytotoxicity.

The liquid of 2-ethoxybenzoic acid cements is composed of 2-ethoxybenzoic acid and eugenol (4-allyl-2-methoxyphenol). Recently, eugenol was reported to produce radicals at a higher pH, which consequently directly damages cells. We examined here whether eugenol radicals are generated from the mixture of eugenol/calcium hydroxide, and also whether 2-ethoxybenzoic acid or acetylsalicylic acid scavenges radicals, using electron spin resonance spectroscopy. Radicals were generated from the mixture of eugenol/calcium hydroxide in 50% dimethylsulfoxide solution. The radical intensity of eugenol in 50% dimethylsulfoxide with 0.1 M sodium bicarbonate buffer (pH 9.5) was dose-dependently reduced by 2-ethoxybenzoic acid, whereas it was enhanced by acetylsalicylic acid. Next, we investigated the cytotoxic effect of eugenol on 2-ethoxybenzoic acid, acetylsalicylic acid, or calcium hydroxide on human pulp fibroblasts or a human submandibular gland cancer cell line. The cytotoxicity of EBA was decreased, whereas that of acetylsalicylic acid was increased by eugenol. In contrast, that of calcium hydroxide was not affected by eugenol. Human pulp fibroblast but not human submandibular gland cells showed a high resistance against calcium hydroxide. The generation of eugenol radicals in the liquid of 2-ethoxybenzoic acid cements caused by oxidation may be suppressed by 2-ethoxybenzoic acid.

Expression of Ca2+/calmodulin-dependent protein kinase (CaMK) Ibeta2 in developing rat CNS.

We observed the onset time and distribution pattern of beta2 isoform of Ca2+/calmodulin-dependent protein kinase I (CaMKIbeta2) in the CNS of the rat during the embryonic period until birth using an immunohistochemical method. The expression of CaMKIbeta2 started at embryological day 10 when the three primary brain vesicles and neural tube are generated from the neural plate. During the embryonic period, highly immunoreactive products were ubiquitously detected in neurons in the CNS, although neurons in the caudate-putamen and globus pallidus were faintly immunostained or immunonegative. High expression of CaMKIbeta2 persisted in the olfactory bulb, lymbic system, neocortex, septal nuclei, amygdala complex, some hypothalamic nuclei, pontine nuclei, Purkinje cells and granule cells in the cerebellar cortex through the developing period. At the subcellular level, CaMKIbeta2 was strongly expressed in nuclei of neurons but faintly in their cytoplasm, suggesting that this protein has an important role in the nuclear signaling pathway. This study demonstrates that expression of CaMKIbeta2 begins at the earliest developmental stage of the rat CNS and persists through the developing period.

Cytotoxic activity of vitamins K1, K2 and K3 against human oral tumor cell lines.

Vitamin K1, K2 and K3 were compared for their cytotoxic activity, radical generation and O2- scavenging activity. Among these compounds, vitamin K3 showed the highest cytotoxic activity against human oral tumor cell lines (HSC-2, HSG), human promyelocytic leukemic cell line (HL-60) and human gingival fibroblast (HGF). Vitamin K3 induced internucleosomal DNA fragmentation in HL-60 cells, but not in HSC-2 or HSG cells. The cytotoxic activity of vitamins K2 and K1 was one and two orders lower, respectively, than K3. Vitamin K2, but not vitamin K3, showed tumor-specific cytotoxic action. ESR spectroscopy showed that only vitamin K3 produced radical(s) under alkaline condition and most potently enhanced the radical intensity of sodium ascorbate and scavenged O2- (generated by hypoxanthine-xanthine oxidase reaction system); vitamin K2 was much less active whereas vitamin K1 was inactive. These data suggest that the cytotoxic activity of vitamin K3 is generated by radical-mediated oxidation mechanism and that this vitamin has two opposing actions (that is, antioxidant and prooxidant), depending on the experimental conditions.

Changes in amino acid pool and utilization during apoptosis in HL-60 cells induced by epigallocatechin gallate or gallic acid.

Recent studies have demonstrated the apoptosis-inducing potential of epigallocatechin gallate (EGCG), a major component of green tea, against various cultured cell lines. By using an amino acid analyzer, we investigated here the possible changes in the amino acid pool and utilization during the apoptosis of HL-60 cells induced by EGCG or gallic acid, a structural unit of tannin. Sublethal concentrations of EGCG initially elevated and then reduced the intracellular concentrations of most of amino acids except for glutamic acid and aspartic acid, whereas lethal concentrations of EGCG continuously reduced these amino acid pools during 6 hours. Both sublethal and lethal concentrations of gallic acid initially elevated and then reduced these amino acid pools. Both inducers elevated the intracellular accumulation and production of arginine and extensively reduced the utilization of other amino acids. These data demonstrate that EGCG showed more severe effects on the amino acid pool and utilization than gallic acid, which may explain, at least in part, the difference in apoptosis-inducing potential between these inducers.

Production of hydrogen peroxide and methionine sulfoxide by epigallocatechin gallate and antioxidants.

Epigallocatechin gallate (EGCG) induced apoptosis-associated characteristics in human oral tumor cell lines more efficiently than ascorbates, gallic acid, vitamin K, flavonoids or steroidal saponins. Since catalase partially inhibited the cytotoxic activity of EGCG, the possible involvement of hydrogen peroxide (H2O2) in cell death induction was investigated, using TCPO chemiluminescence method. Production of H2O2 by EGCG, sodium ascorbate, gallic acid or catechin reached a maximum level within 30 minutes, and was increased up to a plateau level above pH 8. Under optimal conditions, 1 mM EGCG was converted to 1 mM H2O2. At neutral pH, EGCG produced the highest amount of H2O2, followed by gallic acid, sodium ascorbate and catechin. EGCG produced methionine sulfoxide from methionine in the culture medium, while the methionine oxidation by EGCG was significantly reduced in the presence of serum. ESR spectroscopy showed that EGCG, gallic acid and sodium ascorbate, but not catechin, produced radicals under alkaline condition and that all these compounds scavenged superoxide anion, produced by hypoxanthine-xanthine oxidase reaction. EGCG also effectively scavenged the ascorbate and gallate radicals, more efficiently than other compounds. These data suggest that the apoptosis induction by EGCG may be mediated by H2O2 produced in the culture medium.

Cytotoxic activity of benzothiepins against human oral tumor cell lines.

A total of 11 newly synthesized benzothiepins and structurally-related compounds were investigated for cytotoxic activity against both normal and tumor cells. All these compounds showed higher cytotoxic activity against three human oral tumor cell lines (HSC-2, HSC-3, HSG) than against normal human gingival fibroblast (HGF), suggesting tumor-specific cytotoxic action. In general, 3,4-dihydro-1-benzothiepin-5(2H)-ones [1-6] showed higher cytotoxic activity than 2,3-dihydro-1-benzothiepins [7-11]. Compounds 4 (4-bromo-3,4-dihydro-2-(2-oxo-2-phenylethyl)-1-benzothiepin-5(2H)-one), 5 (4-bromo-3,4-dihydro-2-(2-oxopropyl)-1-benzothiepin-5(2H)-one) and 6 (4-bromo-3,4-dihydro-2-[1-(methoxycarbonyl)-1-methylethyl]-1-benzothiepin-5(2H)-one), showed higher cytotoxic activity than compounds 1, 2 and 3, respectively, which had Cl instead of Br at C-4 position. Agarose gel electrophoresis demonstrated that these compounds induced large DNA fragments in oral tumor cells, whereas they produced smear pattern of smaller DNA fragments in human promyelocytic leukemia cells HL-60. These data suggest the medicinal efficacy of benzothiepins.

Relationship between cytotoxic activity and radical intensity of isoflavones from Sophora species.

Among 11 isoflavones tested, genistein [YS13] produced higher cytotoxic activity against human oral tumor cell lines (HSC-2, HSG) than against normal cells (human gingival fibroblast, HGF), suggesting its tumor-specific action. Electron spin resonance (ESR) spectroscopy showed that YS13 did not produce radical, nor scavenged O2*- generated by hypoxanthine-xanthine oxidase reaction system, suggesting that radical-mediated oxidation mechanism is not be involved in the YS13-induced cytotoxicity. Addition of one prenyl group produced YS18 and YS19 with higher anti-Helicobacter pylori activity. Addition of two prenyl groups produced YS21 with the highest cytotoxic activity but lower tumor-specificity. Since YS21 produced the highest amount of radical and most efficiently scavenged O2*-, this compound may induce cytotoxicity by radical-mediated oxidation mechanism. All isoflavones failed to induce anti-human immunodeficiency virus (HIV) activity. These data suggest the medicinal efficacy of isoflavones.

Cytotoxic and DNA damage-inducing activities of low molecular weight phenols from rhubarb.

Six new phenol (anthraquinone or stilbene) glycosides with an acyl group at 6-position of the glucopyranose moiety were isolated from rhubarb (the roots of Rheum palmatum) cultivated in Japan, together with 22 known compounds. Most of these compounds were evaluated for cytotoxic activity against tumor and normal cells and for induction of DNA damage by spore rec-assay. Among them, emodin and aloe-emodin showed higher cytotoxic activities against human oral squamous cell carcinoma (HSC-2) and salivary gland tumor (HSG) cell lines than against normal human gingival fibroblasts (HGF). Chrysophanol 8-O-beta-(6'-acetyl)glucopyranoside, 4-(4'-hydroxyphenyl)-2-butanone 4'-O-beta-D-(2"-O-galloyl-6"-O-cinnamoyl) glucopyranoside, and 6"-O-(4'''-hydroxybenzoyl) resveratroloside exhibited relatively higher cytotoxic activities against all these cells. The other glycosides of anthraquinone or stilbene showed weaker cytotoxic activity against these tumor cell lines, but may be considered as cancer chemopreventive agents. Spore rec-assay with a recombination deficient mutant of Bacillus subtilis M45 demonstrated the DNA damage-inducing activity of emodin and aloe-emodin 15-O-beta-D-glucopyranoside among, rhubarb phenols.