Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore

A previously presented homogeneous assay method, named the excimer-forming two-probe nucleic acid hybridization (ETPH) method, is based on specific excimer formation between two pyrenes attached at the neighboring terminals of two sequential probe oligonucleotides complementary to a single target. In this study, we investigated assay conditions and optimal molecular design of probes for intense excimer emission using a pyrenemethyliodoacetamide-introduced 16mer probe, a pyrene butanoic acid-introduced 16merprobe and a target 32mer. The length of the linker between the pyrene residue and the terminal sugar moiety remarkably influenced the quantum efficiency of excimer emission; the pair of linker arms of these two probes was optimal. The quantum efficiency was also dependent upon the concentrations of dimethylformamide and NaCl added to the assay solution. Spectroscopic measurements and T m analysis showed that an optimal configuration of the two pyrene residues for intense excimer emission might be affected by pyrene-pyrene interaction, pyrene-duplex interaction (intercalation/stacking) and solvent conditions as a whole. We then demonstrated the practicality of the ETPH method with the optimal hybridization conditions thus attained by determining that the concentration of 16S rRNA in extracts from Vibrio mimicus ATCC 33655 cells in exponential growth phase is 18 500 16S rRNA molecules/cell on average.     

Regulation of cell growth-dependent expression of mammalian CDC6 gene by the cell cycle transcription factor E2F

CDC6 of Saccharomyces cerevisiae regulates the DNA replication initiation through the origin recognition complex (ORC). Identification of a human homolog of the CDC6 gene (HsCdc6) suggests a universal role of the gene product in DNA replication. Expression of HsCdc6 is growth-regulated. We investigated the molecular basis of growth-regulated expression of mammalian Cdc6. The promoter activity of isolated HsCdc6 upstream region was activated at late G1 and G1/S boundary in the cell cycle of rat embryonic fibroblast REF52 cells by the addition of serum. The isolated promoter was activated by exogenous expression of E2F without serum stimulation. However a mutant promoter lacking the E2F recognition sites failed to respond to serum stimulation and exogenous expression of E2F. Expression of endogenous Cdc6 was induced by exogenous expression of E2F. Therefore, we concluded that the growth-regulated expression of mammalian Cdc6 was mediated by E2F. Moreover, we demonstrated that exogenous overexpression of either HsCdc6 or HsOrc1 failed to induce DNA synthesis unlike overexpression of E2F1, even though E2F1 induced both Cdc6 and Orc1, suggesting that E2F may regulate the expression of another gene(s), besides Cdc6 and Orc1, required for induction of cellular DNA synthesis in mammalian cells.     

Effects of hypokalaemia on arrhythmogenic risk of quinidine in rats

Plasma potassium concentration plays an important role in the induction of arrhythmia and is closely related to the arrhythmogenicity of various drugs. We quantitatively analyzed the influence of plasma potassium concentration on QT intervals before drug administration and on drug-induced QT prolongation, to estimate the risk of drug-induced arrhythmia under hypokalaemic conditions. The hypokalaemic models were produced by intraperitoneal administration of furosemide and hydrochlorothiazide in male Sprague-Dawley rats. The relationship between the changes in QT intervals and time profiles of plasma quinidine (QND) concentration were analyzed during constant intravenous infusion of QND (10 or 30 mg/kg/h) and post infusion in normal and hypokalaemic rats. The plasma QND concentration reached the therapeutic range (3-7 microg/ml) at the high infusion rate (30 mg/kg/h). No pharmacokinetic differences between normal and hypokalaemic rats were observed. QND induced QT prolongation in parallel with the plasma concentration without hysteresis. Although the potency of QND for QT prolongation was not affected by hypokalaemia, the QT intervals before drug administration were significantly prolonged in hypokalaemic rats (65.90 +/- 1.40 vs 56.60 +/- 0.748 msec, mean +/- SEM, p < 0.0001). Thus, the prolongation of QT intervals before drug administration may act as a risk factor of arrhythmia under hypokalaemic conditions.     

Acoustic damping characterization and microstructure evolution during high-temperature creep of an austenitic stainless steel

We studied the microstructure evolution of an austenitic stainless steel, Type 316L, subjected to tensile creep at 973 K through the monitoring of shear-wave attenuation and velocity using electromagnetic acoustic resonance (EMAR). Contactless transduction based on the Lorentz force mechanism is the key to establishing a monitor for microstructural change in the bulk of metals with high sensitivity. In the short interval, 60 to 70 pct of the creep life, attenuation experiences a peak, independent of the applied stress. A drastic change in dislocation mobility and rearrangement interrupted this novel phenomenon, as is supported by observations using a scanning electron microscope (SEM) and a transmission electron microscope (TEM). The EMAR exhibited the potential for the assessment of damage advance and the prediction of the remaining creep life of metals.     

A study of hypervelocity impact on cryogenic materials

This paper reports a result of hypervelocity impact experiments on cryogenically cooled aluminum alloys and a composite material. Experiments are carried out on a target palate at 122 K. Aluminum spheres at 1.95 km/s in 50 kPa air were impinged against the target plate at cryogenic temperature and the result was compared with room temperature target plates. Hypervelocity impact (HVI) processes were visualized with shadowgraph arrangement and recorded with high-speed video camera and to ensure the temperature dependence we compared HVI tests with metal target plates with AUTODYN 2D and SPH numerical simulations. We found that cryogenic impacts created slight differences of impact damage from room temperature ones, i.e., the shape and averaged diameters of HVI crater holes were less at cryogenic impacts.     

A video DSP with a macroblock-level-pipeline and a SIMD typevector-pipeline architecture for MPEG2 CODEC

A video DSP with macroblock-level-pipeline and a SIMD type vector-pipeline architecture (VDSP2) has been developed, using 0.5 μm triple-layer-metal CMOS technology. This 17.00 mm×15.00 mm chip consists of 2.5 M transistors, and operates at 100 MHz. The real-time encoder and decoder specified in the MPEG2 main profile at the main level can be realized with two VDSP2's and a motion estimation (ME) unit, and one VDSP2 respectively, at an 80 MHz clock rate, with a total power dissipation of 4.2 W at 3.3 V     

Expression of ubiquitin protein in each organ at death from hypothermia

To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-3H]uridine-labeled nucleic acid with 50 microg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 microg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3' to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 microg/ml RNase T1, which preferentially cleaves RNA 3' to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-3H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA.     

Isolation of bioactive compounds from Helix aspersa nerve tissue and the effect of pQPPLPRYamide on heart, esophagus and central neurons of H. aspersa and rectum of Anodonta woodiana

1. Tumour necrosis factor-alpha (TNF-alpha) is implicated in the pathogenesis of many pulmonary and airway diseases. TNF-alpha stimulation may release interleukin-8 (IL-8) in airways mediated via an increase in intracellular oxidant stress. In the present study, we have assessed leukosequestration and IL-8 release in the airways in response to intratracheal administration of human recombinant TNF-alpha, and examined the modulatory role of endogenous NO by pretreatment with a NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). 2. TNF-alpha (10(2)-10(-4) u) was administered intratracheally in male guinea-pigs which were anaesthetized with urethane and were ventilated artificially. TNF-alpha induced a time- and dose-related increase in neutrophil numbers and a concomitant increase in human IL-8 equivalent level retrieved from bronchoalveolar lavage (BAL) with the peak effect at 10(3) u at 6 h of TNF-alpha injection (late phase). Intratracheal administration of recombinant human (rh)IL-8 (0.025, 0.25, 2.5 ng) producing a similar range of human IL-8 equivalent levels in BAL as measured in our results induced neutrophil recovery in BAL fluid to a similar extent. Administration of anti-IL-8 antibody prevented the late phase of neutrophil recruitment induced by TNF-alpha or rhIL-8. 3. Pretreatment with L-NAME significantly enhanced the TNF-alpha (10(3) u)-induced neutrophil recruitment and human IL-8 equivalents production at 6 h, but not at 1 h of TNF-alpha administration (early phase). L-Arginine reversed the responses to L-NAME. Pretreatment with 0.2% DMSO (i.v.) significantly inhibited TNF-alpha-induced neutrophil recruitment and human IL-8 equivalents release both in the early and late phase of the responses. Pretreatment with DMSO also inhibited the enhancement effect of L-NAME on the late phase of TNF-alpha-induced responses. DMSO failed to modify exogenous rhIL-8-induced neutrophil recruitment. Neither L-NAME nor DMSO alone induced any significant change in neutrophil numbers or human IL-8 equivalent level in BAL fluid. 4. Neutrophil depletion by cyclophosphamide pretreatment failed to modify TNF-alpha-induced human IL-8 equivalent release. 5. The expression of beta 2-integrin, CD11b/CD18 on neutrophils was increased only in the late but not early phase of TNF-alpha stimulation. L-NAME failed to modify these responses. 6. In conclusion, we demonstrated that NO may be an important endogenous inhibitor of TNF-alpha-induced leukocyte chemotaxis via inhibition of IL-8 production. Thus, the production of NO in airway inflammatory diseases may play a negative feedback role in self-limiting the magnitude of inflammatory responses.     

Solubility of platinum in molten fluxes as a measure of basicity

The solubility of platinum in the molten BaO-Al₂O₃, BaO-SiO₂, CaO-Al₂O₃, CaO-SiO₂, Na₂O-SiO₂, and CaO-Al₂O₃-SiO₂ systems has been measured in order to seek a new measure of basicity. The solubility of platinum increases with increasing content of basic oxide. The correlations among the solubility of platinum, carbonate, sulfide, and phosphate capacities, basic oxide activities, and theoretical optical basicity are discussed.     

Determination of all elastic constants of orthotropic plate specimens from group velocity data

This paper presents a novel method of nondestructively determining all nine elastic constants of fiber-reinforced orthotropic plate specimens. Group velocities of bulk quasi-longitudinal (QL), pure transverse (PT), and surface skimming pseudo-longitudinal modes are measured in glass-fiber (GF) and carbon-fiber (CF) reinforced Poly Ether Ether Ketone (PEEK) specimens by a point-source/point-receiver (PS/PR) technique. First, the pure index longitudinal moduliC ₁₁,C ₂₂, andC ₃₃, are obtained from the longitudinal (L) group velocities measured in three principal directions. Next, the pure index shear moduli,C ₄₄,C ₅₅, andC ₆₆, are determined by measuring the shear horizontally (SH) polarized PT group velocities in the symmetry planes. Finally, the mixed index elastic constantsC _ij (i j) are calculated from QL group velocities measured in the symmetry planes and using analytic formulas.