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PubMed2004

Purification and characterization of recombinant sTRAIL expressed in Escherichia coli.

As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has drawn considerable attention. This report presented the purification and characterization of soluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized and refolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purified to electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purified sTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with beta-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein.

Apoptosis Regulatory Proteins↗

PubMed2002

Power and coherence of sleep spindle frequency activity following hemispheric stroke.

Brainstem and thalamic structures are known to play a critical role in modulating sleep-wake cycles, but the extent to which the cerebral hemispheres are involved remains unclear. To study the role of the cerebral hemispheres in generating sleep EEG patterns, all-night polysomnographic recordings were collected in subjects with brain damage (n = 30) caused by hemispheric stroke and in hospitalized controls (n = 12). Recordings were made in the acute (< or =10 days post-stroke), subchronic (11-35 days post-stroke) and chronic (>60 days post-stroke) phases of stroke. Bipolar and referential EEG derivations were recorded. Standard sleep stage scoring was conducted using the referential derivation placed opposite the lesion. Sleep stage 2 power and coherence spectra were calculated based on recordings from bipolar derivations. In the mean spectra, the highest spindle frequency peak was identified and its size was calculated relative to the background spectrum. Analysis of visually scored EEG data indicated that, compared with controls, acute phase brain-damaged subjects had lower sleep efficiency and increased waking after sleep onset. The durations of rapid eye movement and non-rapid eye movement sleep stages did not differ significantly between brain-damaged subjects and hospitalized controls. Spectral analyses revealed that, compared with hospitalized controls, brain-damaged subjects had significantly reduced spindle peak sizes in the power and coherence spectra from derivations ipsilateral to the lesion. Within-subject comparisons across time demonstrated that the power and coherence of sleep spindle frequency activity increased significantly from the acute to the chronic phases of stroke, suggesting that plastic mechanisms allowed the possibility of recovery. Our findings provide novel evidence that the cerebral hemispheres are important in generating coherent sleep spindles in humans, and they are consonant with prior empirical and theoretical evidence that corticothalamic projections modulate the generation of synchronous spindle oscillations. Because spindle oscillations are thought to be involved in blocking sensory input to the cortex during sleep, the decrease in synchronous spindle frequency activity following hemispheric stroke may contribute to the observed reduction in sleep continuity.

Adolescent↗

PubMed1990

Base and sequence specificities of aflatoxin B1 binding to single- and double-stranded DNAs.

The inhibitory effect of alfatoxin B1 (AFB1) on the template function for RNA synthesis of several single- and double-stranded DNAs with known base content and sequence was studied in vitro. The results showed that AFB1 strongly inhibits the template function of poly[d(G-C)] and has little, if any, effect on poly[d(A-T)]. Using [3H]AFB1 for the binding, and by spectrum analysis of the appearance of a broad AFB1-DNA adduct peak between 300 and 400 nm right after the typical DNA peak at 260 nm, it is possible to conclude that the binding preference of AFB1 to DNA is: poly[d(G-C)] greater than polydG.polydC greater than polydG greater than polydC, with no detectable binding to poly[d(A-T)]. These studies have therefore provided evidence that the selective inhibition of DNA template function is a direct reflection of the binding specificities of AFB1 to DNA. Furthermore, since there is a 3-fold binding preference of AFB1 for poly[d(G-C)] over polydG.polydC on an equal weight basis, and with very low binding affinity toward either G or C when it is in single-stranded form, these data also suggest: (i) AFB1 binds preferentially to DNA with an alternating G-C sequence compared to DNA with a sequence of contiguous Gs or Cs; and (ii) intercalation may be part of the mechanism for the binding of AFB1 to DNA.

Aflatoxin B1↗

PubMed1996

p53 mutations in hepatocellular carcinoma related to oral contraceptive use.

Oral contraceptives (OCs) are implicated in the development of hepatocellular carcinoma (HCC). Mitogenic stimulation may be the primary mechanism of tumorigenesis, but other factors may also contribute. Mutational spectrum analysis can provide insights into pathogenesis, therefore we analyzed the p53 tumor suppressor gene in 10 HCCs from women with a history of OC use. All were non-Asians whose average OC use was 6.7 years (range 2 months-13 years) and whose mean age at HCC diagnosis was 48.8 years (range 21-67 years). Each tumor was analyzed by immunohistochemistry, DNA sequencing and allelic deletion analysis. Three tumors were positive by p53 immunohistochemistry; allelic deletion analysis identified loss of heterozygosity in one of four informative cases. Two p53 point mutations were found in one tumor containing moderately and well-differentiated components; this patient was negative for all serological markers of hepatitis B and C infections. Both components showed p53 protein accumulation and a GTTval-->GCTala mutation at codon 274. In addition, a silent mutation (ACCthr-->ACTthr) at codon 140 of the p53 gene was detected in the moderately differentiated component of the tumor. These preliminary data indicate that p53 mutations are uncommon in OC-related HCCs. One of the two detected mutations was a G:C-->A:T transition at a non-CpG site, which is characteristic of DNA damage by free radicals. These data support a model whereby estrogens contribute to HCC development primarily through mitogen stimulation and secondarily by mutagenesis via hydroxyl radicals produced during estrogen metabolism. Confirmational analysis of a larger series is warranted.

Adult↗

PubMed1992

Neural regulation of heart rate variability in endurance athletes and sedentary controls.

OBJECTIVE: The aim was to examine the cardiac autonomic responses to orthostatic stress and recovery from steady state exercise in endurance trained athletes and sedentary subjects. METHODS: The power spectrum of heart rate variability was measured before and after exercise in 10 male long distance runners and 14 male sedentary control subjects. Both groups were comparable in sex, age, and body mass index. Continuous ECG recordings were obtained during the following physiological manoeuvres: 45 min supine rest state; 10 min standing; 15 min steady state exercise at 50% maximum workload, and 15 min while supine during post-exercise recovery. The resting heart rate of athletes was lower than controls, at 52(SD 4.9) v 67(8.7) beats.min-1, p < 0.001. Power spectrum analysis was performed using autoregressive modelling. RESULTS: The resting high frequency (HF) vagal component was higher in athletes than controls, at 62 (10.7) v 44(22.4) beats.min-1.Hz-1, p < 0.05. The resting low frequency (LF) peak power was significantly reduced in athletes, at 54(9.9) v 70(19.5) in control, p < 0.05. Although no group differences were observed during upright posture or exercise, the LF:HF area ratio had already returned to pre-exercise levels within 5 min of recovery in athletes. Conversely, it required up to 15 min of recovery before a noticeable decrease in the LF:HF area ratio was seen in controls. CONCLUSIONS: These data support the hypothesis that endurance training modifies heart rate control in whole or in part through neurocardiac mechanisms.

Adult↗

PubMed2006

Atrial fibrillatory rate and sinus rhythm maintenance in patients undergoing cardioversion of persistent atrial fibrillation.

AIMS: The study set out to explore whether an index of atrial electrical electrophysiology can be used to predict atrial fibrillation (AF) relapse, and if the predictive properties differ as a result of arrhythmia duration. METHODS AND RESULTS: The study comprised 175 consecutive patients with persistent AF (median duration 94 days, range 2 to 1044) referred for cardioversion. Twenty-nine patients had arrhythmia duration under 30 days (median 5 days, range 2-26). Atrial fibrillatory rate (AFR) was estimated using a frequency power spectrum analysis of QRST-cancelled ECG. At 1-month follow-up, 56% of the patients had relapsed to AF. The pre-cardioversion mean AFR of those patients was 399+/-52 fibrillations per minute (fpm) compared with 363+/-63 fpm among patients maintaining SR (P<0.0001). In patients with short AF duration, the difference was even more pronounced (424+/-52 vs. 345+/-65 fpm, P<0.01). In this group, a finding of an AFR above the mean value of the study population predicted AF relapse with high accuracy. CONCLUSION: In patients undergoing cardioversion of persistent AF, AF relapse is predicted by a higher AFR. A stronger association is seen in patients with short arrhythmia duration, reflecting either rapid remodelling or pre-existing changes in those who relapse to AF.

Adrenergic beta-Antagonists↗

PubMed2002

Pol32, a subunit of Saccharomyces cerevisiae DNA polymerase delta, suppresses genomic deletions and is involved in the mutagenic bypass pathway.

The Pol32 subunit of S. cerevisiae DNA polymerase (Pol) delta plays an important role in replication and mutagenesis. Here, by measuring the CAN1 forward mutation rate, we found that either POL32 or REV3 (which encodes the Pol zeta catalytic subunit) inactivation produces overlapping antimutator effects against rad mutators belonging to three epistasis groups. In contrast, the msh2Delta pol32Delta double mutant exhibits a synergistic mutator phenotype. Can(r) mutation spectrum analysis of pol32Delta strains revealed a substantial increase in the frequency of deletions and duplications (primarily deletions) of sequences flanked by short direct repeats, which appears to be RAD52 and RAD10 independent. To better understand the pol32Delta and rev3Delta antimutator effects in rad backgrounds and the pol32Delta mutator effect in a msh2Delta background, we determined Can(r) mutation spectra for rad5Delta, rad5Delta pol32Delta, rad5Delta rev3Delta, msh2Delta, msh2Delta pol32Delta, and msh2Delta rev3Delta strains. Both rad5Delta pol32Delta and rad5Delta rev3Delta mutants exhibit a reduction in frameshifts and base substitutions, attributable to antimutator effects conferred by the pol32Delta and rev3Delta mutations. In contrast, an increase in these two types of alterations is attributable to a synergistic mutator effect between the pol32Delta and msh2Delta mutations. Taken together, these observations indicate that Pol32 is important in ensuring genome stability and in mutagenesis.

Canavanine↗

PubMed2000

Auxin-dependent cell wall depositions in the epidermal periplasmic space of graviresponding nodes of Tradescantia fluminensis.

Differential growth of the nodal regions of graviresponding Tradescantia fluminensis (Wandering Jew) was analysed with special respect to the extension-restricting epidermal cells of the opposite growing and growth-inhibited organ flanks. Gravicurvature of horizontally gravistimulated isolated nodes depends on auxin (indolyl-3-acetic acid, IAA) and shows a node-specific profile in which the third node below the tip showed the greatest response. Exogenously supplied gibberellic acid induced no gravitropic growth. Vertically oriented isolated nodes supplied with exogenous IAA showed, on an electron microscopical level, conspicuous membrane invaginations with adjacent wall depositions restricted to the outer tangential epidermal cell walls. Their number was more than doubled by exogenously supplied Ca2+, which inhibited IAA-induced growth. No such changes could be detected in water-incubated segments or inner tissues of IAA-supplied segments. Gravistimulated differential growth of nodes of intact shoots and of nodal segments was characterized by changes similar to the ones induced by exogenous IAA, with greatly increased numbers of wall depositions within the epidermal cells of the growth-inhibited upper organ flank. Similar to the gravistimulated wall depositions, an asymmetric distribution pattern of Ca2+ was detected in the epidermal cell walls employing x-ray energy spectrum analysis (EDX). The results indicate that growth of nodes of Tradescantia fluminensis is regulated via IAA-induced secretion and subsequent infiltration of wall components enabling wall extension. The data support the hypothesis that temporary differential growth during gravicurvature of Tradescantia fluminensis is mediated by the antagonistic effect of Ca(2+)-ions on the infiltration of IAA-induced wall-loosening components into the outer, extension-restricting epidermal walls thereby inhibiting growth.

Calcium↗

PubMed1996

Cry threshold predicts regulatory disorder in newborn infants.

Studied the autonomic regulation of 37 infants with a typical cry threshold and 17 infants with a high cry threshold (typical of problems in nervous system function). Infants with a high cry threshold had a longer latency to cry, a shorter first cry sound, and a shorter overall bout of crying. Spectrum analysis of 2 hours of heart rate variability showed that a high cry threshold was predictive of fewer reliable rhythms and a lower power of the basic 40-min rhythm in heart rate. High cry threshold infants also showed fewer startles and changes in behavioral state. Results suggest a high cry threshold predicts disrupted autonomic regulation and poor coordination among rhythmic systems affecting cardiac activity.

Autonomic Nervous System Diseases↗

PubMed2006

Mutagenicity of UV-irradiated maltol in Salmonella typhimurium.

We investigated the photomutagenicity of maltol (3-hydroxy-2-methyl-4H-pyran-4-one) in bacterial cells. Maltol has a caramel-butterscotch odour and is used as a food additive to impart flavour to bread and cakes. Unirradiated maltol was not mutagenic up to 5 mg/plate in the Ames test. When maltol was irradiated with either UVA (a black light, 320-400 nm, 230 microW/cm(2)) for 5-30 min or UVC (a germicidal lamp, 610 microW/cm(2)) for 3 min in sodium phosphate buffer (pH 7.4) prior to the exposure of bacterial cells, it was mutagenic to Salmonella typhimurium TA100, TA104 and TA97. Mutagenic activation of maltol by UVA-irradiation was more evident in neutral and alkaline conditions (pH 7.0-9.0) than in acidic conditions. On the other hand, photomutagenicity was not observed when maltol was irradiated with UVA in 100 mM NaCl solution or water. The mutagenic photoproduct was stable for at least 60 min after UVA-irradiation. However, addition of thiol compounds (cysteine or glutathione) to the UVA-irradiated maltol diminished the mutagenicity. Mutational spectrum analysis revealed that the predominant base-substitutions induced were G:C-->T:A transversions and G:C-->A:T transitions. An increase of 8-hydroxydeoxyguanosine formation in salmon sperm DNA exposed to maltol and UVA in vitro was detected by HPLC-ECD, but it was too small to explain the photomutagenicity. We are considering the formation of DNA adducts as the photomutagenic mechanism.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1992

Singlet oxygen induced mutation spectrum in mammalian cells.

In order to characterize the molecular nature of singlet oxygen (1O2) induced mutations in mammalian cells, a SV40-based shuttle vector (pi SVPC13) was treated with singlet oxygen arising from the thermal decomposition of the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). After the passage of damaged plasmid through monkey COS7 cells, the vector was shuffled into E. coli cells, allowing the screening of supF mutants. The mutation spectrum analysis shows that single and multiple base substitutions arose in 82.5% of the mutants, the others being rearrangements. The distribution of mutations within the supF gene is not random and some hotspots are evident. Most of the point mutations (98.4%) involve G:C base pairs and G:C to T:A transversion was the most frequent mutation (50.8%), followed by G:C to C:G transversion (32.8%). These results indicate that mutagenesis in mammalian cells, mediated by 1O2-induced DNA damage, is targeted selectively at guanine residues.

Animals↗

PubMed2004

DNA base pair resolution by single molecule force spectroscopy.

The forces that hold complementary strands of DNA together in a double helix, and the role of base mismatches in these, are examined by single molecule force spectroscopy using an atomic force microscope (AFM). These forces are important when considering the binding of proteins to DNA, since these proteins often mechanically stretch the DNA during their action. In AFM measurement of forces, there is an inherent instrumental limitation that makes it difficult to compare results from different experimental runs. This is circumvented by using an oligonucleotide microarray, which allowed a direct comparison of the forces between perfectly matched short oligonucleotides and those containing a single or double mismatch. Through this greatly increased sensitivity, the force contribution of a single AT base pair was derived. The results indicate that the contribution to forces from the stacking interactions is more important than that from hydrogen bonding.

Base Pair Mismatch↗

PubMed2000

Identification of Thermus thermophilus HB8 tRNA (Gm18) methyltransferase gene.

For the purpose of identification of the gene for Thermus thermophilus tRNA (Gm18) methyltransferase [tRNA (guanosine-2'-)-methyltransferase, EC 2.1.1.34], the purified enzyme from native source was analyzed by the peptide-mass mapping. The target gene encoded the amino acid sequences of the obtained peptides was searched in data from Thermus thermophilus HB8 genome-sequencing project. We found the target gene AB05130, which was expected to encode a protein composed of 194 amino acid residues and the molecular mass of this protein was calculated as 22083. The recombinant protein was expressed in E. coli as an active form. The Gm18 formation activity of the purified recombinant protein was confirmed by in vitro methylation followed by two-dimensional thin layer chromatography and Liquid Chromatography/Mass Spectrum analysis of substrate tRNA.

Base Sequence↗

PubMed2000

Light spectral composition in a tropical forest: measurements and model.

I present a simple model that simulates vertical variations in the light spectrum within a forest canopy. The model considers only the vertical, downward transmission of light. The light in each canopy level was assumed to consist of non-intercepted radiation and radiation intercepted within the level and transmitted. The spectrum of non-intercepted light in each canopy level is the same as that of incident light above the canopy (input parameter), whereas the spectrum of transmitted light depends on leaf area index (LAI) and the mean transmission spectrum of leaves. The model was tested in a forest and provided adequate predictions of measured values. Stronger deviations were produced in the near infrared (NIR) waveband in lower canopy levels. Multiple regression between LAI, as the dependent variable, and spectral characteristics (Blue, Green, Red and NIR intensities) had an r(2) of 0.926. As a complement to other methods, I suggest light spectrum analysis as a non-destructive technique for estimating LAI in forest canopies.

Journal Article↗

PubMed1977

Arousal reactions during anesthesia in man.

Power-spectrum analysis of the electroencephalogram and inhaled-exhaled concentrations of halothane (when used), nitrous oxide, and carbon dioxide of 36 patients during surgical operations under general anesthesia were monitored. Electroencephalographic arousal reactions were detected in 24 patients and these were accompanied by irregular respirations in nine patients. Cardiac arrhythmias occurred following an arousal reaction in eight patients. The authors conclude that this electroencephalographic arousal phenomenon occurs frequently and deserves further investigation to define its clinical implications and how it might be avoided.

Adult↗

PubMed1985

EEG quantitation of narcotic effect: the comparative pharmacodynamics of fentanyl and alfentanil.

Fentanyl and alfentanil produce very similar electroencephalographic (EEG) changes in humans. With increasing serum concentrations of either narcotic, progressive slowing in frequency occurs. This narcotic effect on the brain was quantitated using off-line EEG power spectrum analysis. During EEG recording, six unpremedicated patients received a fentanyl infusion (150 micrograms/min), and six received alfentanil (1,500 micrograms/min) until a specific level of EEG depression (delta waves) occurred. Timed arterial blood samples were obtained for measurement of the narcotic serum concentrations. The narcotic-induced EEG changes were found to lag behind (in time) the serum narcotic concentration changes. To accurately relate EEG changes to serum narcotic concentrations, a pharmacodynamic model (inhibitory sigmoid Emax) was combined with a pharmacokinetic model that incorporated an "effect" compartment. (The effect compartment is the separate pharmacokinetic compartment where drug effect is directly proportional to drug concentration. It is the effect site.) The magnitude of the time lag was quantitated by the half-time of equilibration between serum narcotic concentrations and concentrations in the effect compartment. With fentanyl a significantly greater time lag was present (half-time = 6.4 +/- 1.3 min; mean +/- SD) than with alfentanil (half-time = 1.1 +/- 0.3 min). This difference in time lag between blood concentration and effect may be due to the larger brain-blood partition coefficient for fentanyl. The steady-state serum concentration that caused one-half of the maximal EEG slowing was 6.9 +/- 1.5 ng/ml for fentanyl, compared with 520 +/- 163 ng/ml for alfentanil.(ABSTRACT TRUNCATED AT 250 WORDS)

Alfentanil↗

PubMed1986

Power spectrum correlates of changes in consciousness during anesthetic induction with enflurane.

In order to correlate electroencephalogram (EEG) changes during anesthetic induction with level of consciousness, four-channel parasagittal EEG recordings were made during anesthetic induction with enflurane and enflurane-nitrous oxide in oxygen. The EEG was quantitated using power spectrum analysis. Significant EEG changes were identified during all anesthetic inductions; however, the frequency of occurrence of change was significant only during development of amnesia (15 of 20 subjects, P = 0.04). The nature of the EEG changes at this time was agent-specific (P less than 0.05 by chi-square), with high-frequency changes evident in the enflurane group and shifts in amplitude in the 8-12 Hz activity predominating in the nitrous oxide-enflurane group. Anterior dominance could not be documented as a correlate of amnesia or unresponsiveness. The identification of such EEG changes may be valuable in assessing anesthetic depth, but other effects, such as the response of the EEG to surgical stimulation, must be determined before the results are clinically applicable.

Amnesia↗

PubMed1986

Increased sensitivity to etomidate in the elderly: initial distribution versus altered brain response.

To determine the effect of aging on the pharmacokinetics and pharmacodynamics of etomidate, we administered etomidate (5 to 10 mg/min) by intravenous infusion to 21 healthy surgical patients, age 22 to 82 yr. Etomidate produced progressive slowing of the EEG to an easily recognized pattern (stage 3) that determined the dosage endpoint. Subsequent power-spectrum analysis of the EEG gave the median frequency. Median frequency values and simultaneous measurements of blood etomidate concentration were incorporated into a sigmoid Emax pharmacodynamic model that permitted an estimate of IC50, the blood etomidate concentration which produced a 50% reduction in the median frequency. The dose of etomidate required to reach the uniform EEG endpoint decreased significantly with increasing age (r2 = .68) as did the dose needed to produce maximal median frequency depression (r2 = .69). None of the parameters of the pharmacodynamic effect model, including IC50, correlated with age, suggesting that increased brain sensitivity in the elderly does not cause the age-related change in dose requirement. The initial distribution volume for etomidate decreased significantly with increasing age (r = .56), implying that a higher initial blood concentration in the elderly following any given dose of etomidate is part of the cause of the lower dose requirement in the elderly patient. A contracted initial distribution volume in the elderly may result from well described physiologic changes of age. Etomidate clearance also decreased with age. Age-dependent changes in etomidate pharmacokinetics rather than altered brain responsiveness may be the basis for the decreased etomidate dose requirement in the elderly.

Adult↗