Detection of Mg(2+)-dependent endonuclease activity in myeloid leukemia cell nuclei capable of producing internucleosomal DNA cleavage

We detected Mg(2+)-dependent, Ca(2+)-independent endonuclease activity in non-apoptotic myeloid leukemia cell nuclei using autodigestion method which cleaved the chromatin of the autologous leukemia cells to an oligonucleosomal length pattern. Similar endonuclease activity could be successfully recovered in the protein extracts of the human leukemia cell nuclei. The extracts consistently elicited characteristic DNA cleavage of another leukemia cell (KG-1) nuclei as the target, the enzyme activity of which had been inactivated. We propose that this method is a useful tool for the study of endonucleases involved in apoptosis.     

Bepridil reducing levothyroxine-induced enhancement of mitochondria Ca2+ Mg(2+)-ATPase activity in rat cerebrum

AIM: To study if bepridil (Bep) could affect the enhancement of activity of cerebral mitochondria Ca2+ Mg(2+)-ATPase caused by levothyroxine (Lev) in relation to ischemic overload calcium cerebrum injury. METHODS: The experimental hyperthyroidism model with ischemic cerebrum was developed in rats by ig Lev 1 mg.kg-1.d-1 for 7 d. Ca2+ Mg(2+)-ATPase activity and its kinetic parameters were assayed. RESULTS: The activity, Vmax and Km of cerebral mitochondria Ca2+ Mg(2+)-ATPase in control rats were 3.1 +/- 0.8, 5.1 +/- 2.3 mmol.P(i).h-1/g protein and 0.81 +/- 0.08 mmol.L-1 (ATP) respectively, whereas those of hyperthyroid rats were significantly altered to 4.6 +/- 0.5, 8.5 +/- 1.9 mmol.P(i).h-1/g protein and 0.49 +/- 0.11 mmol.L-1 (ATP) respectively. After treated with Bep 10 or 20 mg.kg-1.d-1 ig for 3 d, allabove 3 parameters of the enzyme were very significantly reduced vs those of either control or hyperthyroid. CONCLUSION: Bep, via decreasing Ca2+ Mg(2+)-ATPase activity and increasing the affinity of Ca2+ Mg(2+)-ATPase to ATP, could prevent rat cerebrum from ATP depletion and ischemic overload calcium injury.     

Propranolol and bepridil attenuating levothyroxine-induced rat cardiac hypertrophy and mitochondrial Ca2+ Mg(2+)-ATPase activity elevation

AIM: To study the effects of propranolol and bepridil on levothyroxine-induced rat cardiac hypertrophy and mitochondrial Ca2+ Mg(2+)-ATPase activity elevation. METHODS: Rat heart hypertrophy was induced by i.p., levothyroxine 1 mg.kg-1.d-1 x 10 d. Then rats were treated by ig propranolol (Pro) or bepridil (Bep) 10 mg.kg-1 daily. Ca2+ Mg(2+)-ATPase activity and enzyme kinetic parameters were assayed. RESULTS: The activity and Vmax of mitochondrial Ca2+ Mg(2+)-ATPase isolated from hypertrophic left ventricle were 25 +/- 4 and 35.1 +/- 0.8 mumol Pi.h-1/mg protein, respectively, those of normal were 6.7 +/- 1.8 and 10 +/- 4 mumol Pi.h-1/mg protein, respectively. Apparent K(m) of the hypertrophic group Ca2+ Mg(2+)-ATPase was 0.4 +/- 0.12 mmol.L-1 ATP, and that of normal was 0.59 +/- 0.22 mmol.L-1 ATP. The total protein quantity of hypertrophic left ventricle was 80 +/- 30 mg, and that of normal was 47 +/- 9 mg. After treated with Pro or Bep (both 10 mg.kg-1 ig), the cardiac hypertrophy was attenuated, the enzyme activity and Vmax as well as total protein quantity of hypertrophic left ventricle were reduced to normal level, but apparent K(m) was not affected. CONCLUSION: Both Pro and Bep prevented the myocardium and its mitochondria from ischemia and overload calcium injury.     

Sequence-specific DNA cleavage by Fe2+-mediated fenton reactions has possible biological implications

Preferential cleavage sites have been determined for Fe2+/H2O2-mediated oxidations of DNA. In 50 mM H2O2, preferential cleavages occurred at the nucleoside 5' to each of the dG moieties in the sequence RGGG, a sequence found in a majority of telomere repeats. Within a plasmid containing a (TTAGGG)81 human telomere insert, 7-fold more strand breakage occurred in the restriction fragment with the insert than in a similar-sized control fragment. This result implies that telomeric DNA could protect coding DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and aging. In micromolar H2O2, preferential cleavage occurred at the thymidine within the sequence RTGR, a sequence frequently found to be required in promoters for normal responses of many procaryotic and eucaryotic genes to iron or oxygen stress. Computer modeling of the interaction of Fe2+ with RTGR in B-DNA suggests that due to steric hindrance with the thymine methyl, Fe2+ associates in a specific manner with the thymine flipped out from the base stack so as to allow an octahedrally-oriented coordination of the Fe2+ with the three purine N7 residues. Fe2+-dependent changes in NMR spectra of duplex oligonucleotides containing ATGA versus those containing AUGA or A5mCGA were consistent with this model.     

A Mg(2+)-dependent endonuclease is responsible for internucleosomal DNA fragmentation in human B lymphoblastic IM9 cells

We have identified a Mg(2+)-dependent endonuclease activity from human B lymphoblastic IM9 cell lysates and nuclei using autodigestion method and DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay system. The level of the endonuclease activity in cell lysates was significantly decreased at certain stage by treatment of the cells with cycloheximide. However, the enzyme activity consistently remained for over 12 hours in the isolated nuclei of the apoptotic IM9 cells. The Mg(2+)-dependent endonuclease isolated from the nuclei by native-PAGE elution was able to catalyze the conversion of supercoiled plasmid DNA into linear form. This particular endonuclease activity was not detected in cycloheximide treated-U937 cells. Several lines of experimental evidence suggest that the Mg(2+)-dependent endonuclease localized in the nucleus may be responsible for the DNA fragmentation of apoptotic IM9 cells.     

Significant enhancement of blue photoluminescence intensity of Dy~(3+) modified Sr_6Ca_4(PO_4)_6F_2:Eu~(2+) phosphors

Blue-emitting phosphors Sr_6 Ca_4(PO_4)_6 F_2:Eu~(2+)(SCPF:Eu~(2+)),Sr_6 Ca_4(PO_4)_6 F_2:Eu~(2+),Dy~(3+)(SCPF:Eu~(2+),Dy~(3+))and Sr_6 Ca_4(PO_4)_6 F_2:Eu~(2+),Dy~(3+),Si~(4+)(SCPF:Eu~(2+),Dy~(3+),Si~(4+)) with apatite structure were successfully synthesized by traditional solid-state reaction under reducing atmosphere.Eu~(2+),Dy~(3+) and Si4+ions occupy the corresponding sites of Sr~(2+),Ca~(2+) and P~(5+).Strong broad blue photo luminescence band is exhibited in SCPF:Eu~(2+),Dy~(3+) phosphor ranging from 400 to 550 nm centered at 455 nm and Dy~(3+) ions are vital in creating traps.Emission intensity of Eu~(2+),Dy~(3+) co-doped SCPF:0.02 Eu~(2+),0.02 Dy~(3+) is about 1.8 times that of SCPF:0.02 Eu~(2+) and electron trap centers serve as energy transporting media.To further elucidate the formation and effect of the specific defect on the luminescence of SCPF:0.02 Eu~(2+),0.02 Dy~(3+) phosphor,the thermoluminescence properties,decay curves and thermal stability studies were performed while the Si~(4+)-P~(5+) charge compensated pho sphor SCPF:0.02 Eu~(2+),0.02 Dy~(3+),0.02 Si~(4+) was prepared as a contrast.All the results of present work indicate that Dy~(3+) co-doping can obviously enhance photoluminescence intensity of SCPF:0.02 Eu~(2+) by the electron traps generated by non-equivalence replacement of Dy~(3+)-Ca~(2+).     

Broadband Cr~(3+)-sensitized upconversion luminescence of LiScSi_2O_6:Cr~(3+)/Er~(3+)

Broadband sensitization is an effective strategy to enhance the upconversion luminescence(UCL) of lanthanide ions.Herein,novel UC materials LiScSi_2 O_6:Cr~(3+)/Er~(3+)(LSS:Cr~(3+)/Er~(3+)) were synthesized by high-temperature solid state reaction and their luminescent properties were investigated.LSS:Cr~(3+)/Er~(3+)has the broadband absorption in the spectral range of 600-800 nm,and meanwhile shows green UC emissions of Er~(3+)upon pumping Cr~(3+) by the 690 nm laser.The UCL of LSS:Cr~(3+)/Er~(3+)belongs to the twophoton process and is attributed to the energy transfer upconversion mechanism.The effects of the Cr~(3+)and Er~(3+)concentration as well as the Yb~(3+)introduction were also studied.LSS:Cr~(3+)/Yb~(3+)/Er~(3+) exhibits the interesting dual-mode UCL,capable of generating the UCL of Cr~(3+) upon pumping Yb~(3+)ions and the UCL of upon pumping Cr~(3+) ions.This research might promote the development of novel broadband Cr~(3+)-sensitized UC materials.     

The biological relevance of the binding of calcium ions by inositol phosphates

The binding of Ca2+ (chelation) by myo-inositol polyphosphates at pH 7.0 was studied using a Ca(2+)-sensitive electrode. Glucose 6-phosphate (used as a model for a monophosphate) bound Ca2+ with an affinity of 152 +/- 31 liters/mol and a molar ratio of 0.94 +/- 0.02. Inositol 3,4-bisphosphate, inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol hexakisphosphate showed affinities of 9.0 +/- 2.1 x 10(3), 6.3 +/- 1.5 x 10(3), 6.2 x 10(4), and 1.92 +/- 0.47 x 10(5) liters/mol, respectively, and molar ratios of 0.92 +/- 0.49, 0.95 +/- 0.10, 0.75, and 2.5 +/- 0.5. In general, the affinity increased with the number of phosphate substituents on the inositol ring, although the stereochemistry is also expected to be important. This suggests that for the physiologically relevant inositol phosphates (tris-, tetrakis-, pentakis-, and hexakis-) half-maximal Ca2+ binding will occur in the Ca2+ concentration range of approximately 5 x 10(-6) to 2 x 10(-4) M. This range lies between the basal intracellular and the fee extracellular Ca2+ levels (10(-7) and 10(-3) M), respectively, and may therefore be of physiological importance. Chelation provides a possible simple explanation for the inhibition by Ca2+ of inositol 1,4,5-trisphosphate binding to its receptor in rat cerebellum and other tissues. It may also have a role in limiting inositol phosphate-mediated increases in intracellular Ca2+.     

Mg(2+)-dependent inhibition of KATP by sulphonylureas in CRI-G1 insulin-secreting cells

1 Patch-clamp recording techniques were used to examine the effects of tolbutamide, glibenclamide, meglitinide and thiopentone on KATP in CRI-GI insulin-secreting cells in the presence and absence of Mg2+. 2 In the absence of Mg2+ in the intracellular bathing solution, tolbutamide was significantly less effective when applied either to the intracellular or to the extracellular surfaces of cell-free patches. Removal of extracellular Mg2+ did not alter the effectiveness of tolbutamide provided that Mg2+ was present at the intracellular surface of the patch. 3 Tolbutamide was also significantly less effective when applied to the intracellular surface of cell-free patches when Mn2+ was used as a replacement for Mg2+. 4 Both the sulphonylurea, glibenclamide and the non-sulphonylurea derivative, meglitinide also showed Mg2+ dependent inhibitory effects in cell-free patches. In contrast, the barbiturate thiopentone inhibited KATP in a Mg(2+)-independent manner. 5 Whole-cell IK(ATP) were used to quantify the effects of tolbutamide and glibenclamide in the presence and absence of intracellular Mg2+. Concentration-inhibition curves, in the presence of intracellular Mg2+, resulted in IC50 values of 12.1 microM and 2.1 nM for tolbutamide and glibenclamide, respectively. In the absence of intracellular Mg2+, the corresponding IC50 values were 25.3 mM and 3.6 microM, respectively. The values of IC50 for thiopentone in the presence and absence of intracellular Mg2+ were 69.4 microM and 69.2 microM, respectively. 6 With respect to the high affinity binding sites for [3H]-glibenclamide in CRI-G1 membranes, no significant differences were found between the dissociation constants for, or the maximal binding capacities of, [3H]-glibenclamide in the presence or absence of Mg2+. 7. In the CRI-G1 insulin-secreting cell line, it is concluded that intracellular Mg2+ does not influence the affinity of the sulphonylureas for the sulphonylurea receptor but that this ion is critically important for the interaction between the sulphonylurea receptor and KATP.     

Decreased duration of Ca(2+)-mediated plateau potentials in striatal neurons from aged rats

1. The influence of age on striatal neuron Ca2+ physiology was studied through an analysis of intracellularly recorded Ca(2+)-mediated plateau potentials. In vitro brain slices from young and aged rats were treated with the K+ channel blocker tetraethylammonium (30 mM) to facilitate the expression of plateau potentials. A sample of neurons was also filled with biocytin and post hoc correlations were performed between morphology and physiology. 2. Testing of sampling parameters in neurons from young rats revealed that tetrodotoxin did not affect the amplitude or duration of plateau potentials. The membrane potential induced during plateau testing and the rate of plateau potential generation, however, had to be held constant because these variables affected plateau potential duration. 3. A significant age-related decrease was found in the duration of Ca(2+)-mediated plateau potentials that could not be explained by alterations in the activation or inactivation properties of the plateau potential. Investigation into relationships between cell morphology and plateau potential duration revealed a number of correlations. Soma size and dendritic length were correlated with plateau potential duration, independent of age (hierarchical regression), and an age-related decrease in dendritic length but not in soma size was found. Spine density and plateau potential duration were also correlated, but the significance depended on the variance associated with age. These data indicate that the extent of somadendritic membrane (including spines) affects plateau potential duration in striatal neurons and that dendrite and spine loss in aged animals may contribute to age-related decreases in plateau potential duration. 4. The response to replacement of Ca2+ with Ba2+ was age dependent, with Ba2+ causing a greater increase in the duration of plateau potentials in young neurons. These data rule out an increase in Ca(2+)-mediated inactivation of Ca2+ channels as a primary cause for the shortening of plateau potentials in aged neurons. Our morphological findings suggest that dendritic regression in aged neurons may have reduced the number of Ca2+ channels participating in plateau potential generation, but other mechanisms related to changes in the type of Ca2+ channel expressed and possible differences in their inactivation kinetics may also contribute to the age-related change in plateau potential duration.     

Taurine regulation of Ca2+ uptake and (Ca(2+)+Mg2+)-ATPase in developing chick B-cells

1. The objective of the present study was to determine the effect of age and taurine on chick B cell calcium uptake and membrane (Ca(2+)+Mg2+)-ATPase activity in 1-4-week-old chicks. 2. The calcium uptake rate decreased with age (P < 0.05) and was further decreased by taurine (P < 0.05). 3. (Ca(2+)+Mg2+)-ATPase activity increased with age (P < 0.05) and was stimulated by taurine (P < 0.05). 4. The data demonstrate that the flux of calcium across the B-cell membrane changes during early post-hatch development, and that taurine regulates both the influx and efflux of calcium in chick B-cells.     

Sperm-egg fusion in the sea urchin is blocked in Mg(2+)-free seawater

Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2+ spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al., Dev. Growth Differ. 22, 531-41, 1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2+ to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+ concentration of the egg was observed after insemination in the absence of Mg2+, although both could be observed after the addition of Mg2+. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2+ are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane.     

Effect of Gd~(3+)-,Pr~(3+)-or Sm~(3+)-substituted cobalt-zinc ferrite on photodegradation of methyl orange and cytotoxicity tests

Gd~(3+)-,Pr~(3+)-or Sm~(3+)-doped Co-Zn(Co_(0.5)Zn_(0.5)Fe_2 O_4) magnetic ferrites(i.e.,Co_(0.5)Zn_(0.5)Gd_(0.1)Fe_(1.9)O_4,Co_(0.5)Zn_(0.5)Pr_(0.1)Fe_(1.9)O_4 and Co_(0.5)Zn_(0.5)Sm_(0.1)Fe_(1.9)O_4) were prepared using a facile sol-gel approach,and the structure,surface morphology and chemical composition of the products were studied by means of scanning electron microscopy(SEM),energy dispersive X-ray analysis(EDX),X-ray diffraction(XRD),UVvisible diffuse reflectance spectroscopy(DRS),photoluminescence(PL) spectroscopy,Fourier transform infrared spectroscopy(FT-IR) and vibrating sample magnetometer(VSM) spectroscopy.XRD patterns show the Co-Zn product is composed of cubic spinel phases with few impurities or secondary phases,and the average crystallite sizes of the samples are determined to be approximately～51—80,～99—181,～68—103 and～83—133 nm.Also the coercivity and remnant and saturation magnetizations,evaluated by vibrating sample magnetometer(VSM),are found to increase linearly with the incorporation of Gd3+,Pr3+and Sm3+in the product formulation.The CO_(1-x)Zn_xFe_(2-y)R_yO_4 photocatalyst sample is found to display a red shift in its absorption,and exhibits outstanding photocatalytic effects in the degradation of MO under ultraviolet(UV) light.This is attributed to the reduction of the band gap of cobalt-zinc ferrite due to the presence of rare earth ions.Further in vitro evaluations of the cytotoxic effects of the synthesized nanoparticles were performed on a HeLa cell line.     

Preparation and photocatalytic activity of La~(3+) and Eu~(3+) co-doped TiO_2 nanoparticles: photo-assisted degradation of methylene blue

Rare earth ions La3+ and Eu3+ co-doped TiO2 photocatalyst (La-Eu/TiO2) was prepared by sol-gel method, and characterized by various techniques such as X-ray diffraction (XRD), specific surface area and porosity (BET and BJH), scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), UV-vis diffuse reflectance spectroscopy (DRS) and X-ray photoelectron spectroscopy (XPS). The photocatalytic activity of the La-Eu/TiO2 was evaluated by the degradation of methylene blue (MB) ...     

Luminescence of Eu~(2+) and Eu~(2+)-Mn~(2+) in sodium scandium diphosphate NaScP_2O_7 crystal

In this work, the photoluminescence(PL) of NaScP_2O_7:Eu~(2+) and NaScP_2O_7:Eu~(2+),Mn~(2+) was investigated. Phase purity was checked using X-ray powder diffractometry(XRD). PL excitation and emission spectra were recorded to elucidate the PL properties of NaScP_2O_7:Eu~(2+) and NaScP_2O_7:Eu~(2+),Mn~(2+). Furthermore, fluorescence lifetime measurements were performed. PL and lifetime measurements were carried out from 10 to 525 K. Moreover, the Eu~(2+) site occupation was discussed. It turned out that the incorporated Eu~(2+) ions substituted for Na+ site and occupied two different sites. Temperature dependent PL measurements indicated the emission intensity decreased with increasing temperature due to temperature quenching in NaScP_2O_7:Eu~(2+). Fluorescence lifetimes of Eu~(2+) in NaScP_2O_7:Eu~(2+) almost did not change with a decay constant τ=~0.53 μs in the temperature range of 10–280 K, and then shortened due to temperature quenching. The luminescent lifetime reached ~0.05 μs at T=525 K. Finally, it was found that energy transfer occurred from Eu~(2+) to Mn~(2+) in co-doped NaScP_2O_7:Eu~(2+),Mn~(2+).