Distribution of stable DnaA-binding sites on the Bacillus subtilis genome detected using a modified ChIP-chip method.

We developed a modified ChIP-chip method, designated ChAP-chip (Chromatin Affinity Precipitation coupled with tiling chip). The binding sites of Bacillus subtilis Spo0J determined using this technique were consistent with previous findings. A DNA replication initiator protein, DnaA, formed stable complexes at eight intergenic regions on the B. subtilis genome. Characterization of the binding sequences suggested that two factors -- the local density of DnaA boxes and their affinities for DnaA -- are critical for stable binding. We further showed that in addition to autoregulation, DnaA directly modulate the expression of sda in a positive, and ywlC and yydA in a negative manner. Examination of possible stable DnaA-binding sequences in other Bacillus species suggested that DnaA-dependent regulation of those genes is maintained in most bacteria examined, supporting their biological significance. In addition, a possible stable DnaA-binding site downstream of gcp is also suggested to be conserved. Furthermore, potential DnaA-binding sequences specific for each bacterium have been identified, generally in close proximity to oriC. These findings suggest that DnaA plays several additional roles, such as control of the level of effective initiator, ATP-DnaA, and/or stabilization of the domain structure of the genome around oriC for the proper initiation of chromosome replication.     

Effect of polyanions and polycations on adenosine 3',5'-monophosphate-binding and protein kinase activity.

Histone, protamine, poly-L-arginine, and poly-L-lysine enhance the binding of adenosine 3′,5′-monophosphate (cyclic AMP) to rat liver cyclic AMP-dependent protein kinase as determined by Millipore filtration assay. Poly-L-glutamic acid and poly-L-aspartic acid suppress cyclic AMP-binding stimulated by histone. Poly-L-glutamic acid and poly-L-aspartic acid are effective against protein kinase and result in decrease in initial reaction velocity when histone is used as a protein substrate. Incubation of cyclic AMP-dependent protein kinase with 6 μg poly-L-glutamic acid produces half-maximal inhibition of cyclic AMP-dependent protein kinase when 30 μg histone is used as substrate.     

Hereditary respiration deficiency in Saccharomycodes ludwigii

Saccharomycodes ludwigii, supposed to be petite-negative, gave rise to respiration-deficient mutants when acriflavine and ultraviolet irradiation, respectively, were applied to this yeast, strain IFO 1194. The frequency of such mutants was very low as compared with that in Saccharomyces cerevisiae and other petite-positive yeasts. Cytochrome composition was characterized by spectrophotometry at the temperature of liquid nitrogen. The respiratory mutants examined contained cytochrome c unaltered in quality and quantity. Cytochrome b was often present only in small amounts though never absent, while cytochrome a+a₃ was either present or absent. The respiratory mutants could form zygotes after conjugation with a wild-type culture of opposite mating type ( vs. a). The hybridization and segregation analysis of spore tetrads showed the inheritance of respiratory mutant character to be either Mendelian or non-Mendelian and similar to that of pet (nuclear) and rho^- (cytoplasmic) mutants, respectively, in Saccharomyces cerevisiae.     

Delayed sleep/wake rhythm and excessive daytime sleepiness correlate with decreased daytime brain activity during cognitive task in university students

Insufficient sleep and irregular sleep/wake rhythm are common problems among university students. We investigated the effect of sleep/wake rhythm and excessive daytime sleepiness (EDS) on the cortical oxygenation as measured by near-infrared spectroscopy (NIRS) and cognitive performance in university students. Peak- and integral values by a word fluency task were measured with NIRS. EDS was evaluated by the Epworth sleepiness scale (ESS), and performance function was evaluated using N-back task. Peak cerebral oxygenation was significantly correlated with ESS, bedtime, wake-up time, and median time of sleep. Accuracy on 2-back task was significantly correlated with integral value. Peak- and integral values were significantly lower, and bedtime and median time of sleep were significantly delayed in the EDS group than in the non-EDS group. EDS accompanied by delayed sleep/wake rhythm and short sleep duration may play an important role in decreasing daytime brain activity and cognitive performance.     

Electrical recognition of label-free oligonucleotides upon streptavidin-modified electrode surfaces

For the purpose of developing a direct label-free electrochemical detection system, we have systematically investigated the electrochemical signatures of each step in the preparation procedure, from a bare gold electrode to the hybridization of label-free complementary DNA, for the streptavidin-modified electrode. For the purpose of this investigation, we obtained the following pertinent data; cyclic voltammogram measurements, electrochemical impedance spectra and square wave voltammogram measurements, in Fe(CN)₆ ³⁻/Fe(CN)₆ ⁴⁻ solution (which was utilized as the electron transfer redox mediator). The oligonucleotide molecules on the streptavidin-modified electrodes exhibited intrinsic redox activity in the ferrocyanide-mediated electrochemical measurements. Furthermore, the investigation of electrochemical electron transfer, according to the sequence of oligonucleotide molecules, was also undertaken. This work demonstrates that direct label-free oligonucleotide electrical recognition, based on biofunctional streptavidin-modified gold electrodes, could lead to the development of a new biosensor protocol for the expansion of rapid, cost-effective detection systems.     

Variation in metal concentrations in the brown alga Undaria pinnatifida in Osaka Bay, Japan

In order to evaluate the usefulness of a biomonitoring system using seaweeds for assessing the geographic distribution of metal ions in coastal seawaters, the metal concentrations in the sporophytes of an annual kelp, Undaria pinnatifida (Harvey) Suringar, were collected at 15 localities in Osaka Bay, Japan and compared. About 160 cm² of the blade was cut out from the central part of clean sporophytes, rinsed in filtered seawater using an ultrasonic cleaning bath, and freeze‐dried. After digestion with 12% HNO₃ in a microwave apparatus, metal concentrations in the samples were determined by inductively coupled plasma mass spectrometry. The concentrations (dry weight basis) of most examined elements were in the parts per million range; Cr, 0.48–3.18; Ni, 0.77–5.94; Cu, 3.20–43.8; Zn, 11.3–86.8; Pb, 0.14–3.53. Comparisons of metal compositions of the U. pinnatifida samples from the northeastern area of the bay, which has a large urban population and highly developed industries inland, showed high concentrations of Cd, Pb and Cu compared with the samples from the southwestern area of the bay where the population and industries are much smaller. This suggests that U. pinnatifida metal loads can be used as a marker to track the geographic distributions of the metal concentrations in coastal seawaters, reflecting inland human activities such as shipbuilding and repairing in port areas, and can be used as a useful biomonitoring system of coastal environments for long‐term trend.     

18F-labeled flavones for in vivo imaging of β-amyloid plaques in Alzheimer’s brains

In vivo imaging of β-amyloid (Aβ) aggregates in the brain may lead to early detection of Alzheimer’s disease (AD) and monitoring of the progression and effectiveness of treatment. The purpose of this study was to develop novel ¹⁸F-labeled amyloid-imaging probes based on flavones as a core structure. Fluoropegylated (FPEG) flavone derivatives were designed and synthesized. The affinity of the derivatives for Aβ aggregates varied from 5 to 321 nM. In brain sections of AD model mice, FPEG flavones with the dimethylamino group intensely stained β-amyloid plaques. In biodistrubution experiments using normal mice, they displayed high uptake in the brain ranging from 2.9 to 4.2%ID/g at 2 min postinjection. The radioactivity washed out from the brain rapidly (1.3–2.0%ID/g at 30 min), which is highly desirable for β-amyloid imaging agents. FPEG flavones may be potential PET imaging agents for β-amyloid plaques in Alzheimer’s brains.     

16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel Electrophoresis

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.     

Overexpression of cdk4/cyclin D1 induces apoptosis in PC12 cells in the presence of trophic support.

The induction of apoptosis by cell cycle regulator molecules under conditions optimal for exponential growth was examined in rat pheochromocytoma PC12 cells by overexpression of cyclins and cyclin-dependent kinases (cdks). By flow cytometry and by immunofluorescence, only cells overexpressing cdk4 or cyclin D1 underwent apoptosis, which was not associated with G1-arrest. Cdk4 kinase activity was significantly higher in cdk4-, or cyclin D1-expressing cells. Furthermore, induction of apoptosis by cdk4 was abrogated by co-transfection of p16(INK4), or dominant negative cdk4. These results suggest that upregulation of cdk4 kinase activity is a primary and critical mediator of apoptosis in PC12 cells under physiological conditions.     

Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes.

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor (TLR) 4, a member of the TLR family that participates in pathogen recognition. TLRs recruit a cytoplasmic protein, MyD88, upon pathogen recognition, mediating its function for immune responses. Two major pathways for LPS have been suggested in recent studies, which are referred to as MyD88-dependent and -independent pathways. We report in this study the characterization of the MyD88-independent pathway via TLR4. MyD88-deficient cells failed to produce inflammatory cytokines in response to LPS, whereas they responded to LPS by activating IFN-regulatory factor 3 as well as inducing the genes containing IFN-stimulated regulatory elements such as IP-10. In contrast, a lipopeptide that activates TLR2 had no ability to activate IFN-regulatory factor 3. The MyD88-independent pathway was also activated in cells lacking both MyD88 and TNFR-associated factor 6. Thus, TLR4 signaling is composed of at least two distinct pathways, a MyD88-dependent pathway that is critical to the induction of inflammatory cytokines and a MyD88/TNFR-associated factor 6-independent pathway that regulates induction of IP-10.