ATM Regulates Adipocyte Differentiation and Contributes to Glucose Homeostasis.

Ataxia-telangiectasia (A-T) patients occasionally develop diabetes mellitus. However, only limited attempts have been made to gain insight into the molecular mechanism of diabetes mellitus development in A-T patients. We found that Atm-/- mice were insulin resistant and possessed less subcutaneous adipose tissue as well as a lower level of serum adiponectin than Atm+/+ mice. Furthermore, in vitro studies revealed impaired adipocyte differentiation in Atm-/- cells caused by the lack of induction of C/EBPα and PPARγ, crucial transcription factors involved in adipocyte differentiation. Interestingly, ATM was activated by stimuli that induced differentiation, and the binding of ATM to C/EBPß and p300 was involved in the transcriptional regulation of C/EBPα and adipocyte differentiation. Thus, our study sheds light on the poorly understood role of ATM in the pathogenesis of glucose intolerance in A-T patients and provides insight into the role of ATM in glucose metabolism.

A new microcolumn-type microchip for examining the expression of chimeric fusion genes using a nucleic acid sandwich hybridization technique.

We report a new type of microcolumn installed in a microchip. The architecture allows use of a nucleic acid sandwich hybridization technique to detect a messenger RNA (mRNA) chain as a target. Data are presented that demonstrate that the expression of a chimeric fusion gene can be detected. The microcolumn was filled with semi-transparent microbeads made of agarose gel that acted as carriers, allowing increased efficiency of the optical detection of fluorescence from the microcolumn. The hybrid between the target trapped on the microbeads and a probe DNA labeled with a fluorescent dye was detected by measuring the intensity of the fluorescence from the microcolumn directly. These results demonstrate an easy and simple method for determining the expression of chimeric fusion genes with no preamplification.

Noninvasive evaluation of contractile behavior of cardiomyocyte monolayers based on motion vector analysis.

A noninvasive method for the characterization of cardiomyocyte contractile behavior is presented. Light microscopic video images of cardiomyocytes were captured with a high-speed camera, and motion vectors (which have a velocity dimension) were calculated with a high spatiotemporal resolution using a block-matching algorithm. This method could extract contraction and relaxation motions of cardiomyocytes separately and evaluate characteristics such as the beating rate, orientation of contraction, beating cooperativity/homogeneity in the monolayer, and wave propagation of impulses. Simultaneous phase-contrast imaging and calcium (Ca2+) fluorescence measurements confirmed that the timing of the maximum shortening velocity of cardiomyocytes correlated well with intracellular Ca2+ transients. Based on our analysis, gap junction inhibitors, 1-heptanol (2 mM) or 18-ß-glycyrrhetinic acid (30 µM), resulted in clear changes in beating cooperativity and the propagation pattern of impulses in the cardiomyocyte monolayer. Additionally, the time dependence of the motion vector length indicated a prolonged relaxation process in the presence of potassium (K+) channel blockers, dl-sotalol (1 µM), E-4031 (100 nM), or terfenadine (100 nM), reflecting the prolonged QT (Q wave and T wave) interval of cardiomyocytes. Effects of autonomic agents (acetylcholine or epinephrine [EPI]) or EPI and propranolol on cardiomyocytes were clearly detected by the alterations of beating rate and the motion vector length in contraction and relaxation processes. This method was noninvasive and could sensitively evaluate the contractile behavior of cardiomyocytes; therefore, it may be used to study and/or monitor cardiomyocyte tissue during prolonged culture periods and in screens for drugs that may alter the contraction of cardiomyocytes.

Fabrication of asymmetric molecular junctions by the oriented assembly of dithiocarbamate rectifiers.

The oriented assembly of molecules on metals is a requirement for rectification in planar metal-molecule-metal junctions. Here, we demonstrate how the difference in adsorption kinetics between dithiocarbamate and thioacetate anchor groups can be utilized to form oriented assemblies of asymmetric molecules that are bound to Au through the dithiocarbamate moiety. The free thioactate group is then used as a ligand to bind Au nanoparticles and to form the desired metal-molecule-metal junction. Besides allowing an asymmetric coupling to the electrodes, the molecules exhibit an asymmetric molecular backbone where the length of the alkyl chains separating the electrodes from a central, para-substituted phenyl ring differs by two methylene units. Throughout the junction fabrication, the layers were characterized by photoelectron spectroscopy, infrared spectroscopy, and scanning tunneling microscopy. Large area junctions using a conducting polymer interlayer between a mercury-drop electrode and the self-assembled monolayer prove the relationship between electrical data and molecular structure.

Point-of-care testing system enabling 30 min detection of influenza genes.

We developed a portable and easy-to-use nucleic acid amplification test (NAT) system for use in point-of-care testing (POCT). The system shows sensitivity that is sufficiently higher than that of the currently available rapid diagnostic kit and is comparable to that of real-time reverse transcription polymerase chain reaction (RT-PCR) for influenza testing.

Dielectric coagulometry: a new approach to estimate venous thrombosis risk.

We present dielectric coagulometry as a new technique to estimate the risk of venous thrombosis by measuring the permittivity change associated with the blood coagulation process. The method was first tested for a simple system of animal erythrocytes suspended in fibrinogen solution, where the coagulation rate was controlled by changing the amount of thrombin added to the suspension. Second, the method was applied to a more realistic system of human whole blood, and the inherent coagulation process was monitored without artificial acceleration by a coagulation initiator. The time dependence of the permittivity at a frequency around 1 MHz showed a distinct peak at a time that corresponds to the clotting time. Our theoretical modeling revealed that the evolution of heterogeneity and the sedimentation in the system cause the peak of the permittivity.

Noninvasive method for assessing the human circadian clock using hair follicle cells.

A thorough understanding of the circadian clock requires qualitative evaluation of circadian clock gene expression. Thus far, no simple and effective method for detecting human clock gene expression has become available. This limitation has greatly hampered our understanding of human circadian rhythm. Here we report a convenient, reliable, and less invasive method for detecting human clock gene expression using biopsy samples of hair follicle cells from the head or chin. We show that the circadian phase of clock gene expression in hair follicle cells accurately reflects that of individual behavioral rhythms, demonstrating that this strategy is appropriate for evaluating the human peripheral circadian clock. Furthermore, using this method, we indicate that rotating shift workers suffer from a serious time lag between circadian gene expression rhythms and lifestyle. Qualitative evaluation of clock gene expression in hair follicle cells, therefore, may be an effective approach for studying the human circadian clock in the clinical setting.

Detection of the CLOCK/BMAL1 heterodimer using a nucleic acid probe with cycling probe technology.

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), has enabled rapid acquisition of genomic information. Here we report an analogous technique for the detection of an activated transcription factor, a transcription element-binding assay with fluorescent amplification by apurinic/apyrimidinic (AP) site lysis cycle (TEFAL). This simple amplification assay can detect activated transcription factors by using a unique nucleic acid probe containing a consensus binding sequence and an AP site, which enables the CPT reaction with AP endonuclease. In this article, we demonstrate that this method detects the functional CLOCK/BMAL1 heterodimer via the TEFAL probe containing the E-box consensus sequence to which the CLOCK/BMAL1 heterodimer binds. Using TEFAL combined with immunoassays, we measured oscillations in the amount of CLOCK/BMAL1 heterodimer in serum-stimulated HeLa cells. Furthermore, we succeeded in measuring the circadian accumulation of the functional CLOCK/BMAL1 heterodimer in human buccal mucosa cells. TEFAL contributes greatly to the study of transcription factor activation in mammalian tissues and cell extracts and is a powerful tool for less invasive investigation of human circadian rhythms.

Contrast improvement of terahertz images of thin histopathologic sections.

We present terahertz images of 10 µm thick histopathologic sections obtained in reflection geometry with a time-domain spectrometer, and demonstrate improved contrast for sections measured in paraffin with water. Automated segmentation is applied to the complex refractive index data to generate clustered terahertz images distinguishing cancer from healthy tissues. The degree of classification of pixels is then evaluated using registered visible microscope images. Principal component analysis and propagation simulations are employed to investigate the origin and the gain of image contrast.

Pharmacological effects of Hachi-mi-jio-gan extract (Harncare) on the contractile response and on pharmacologically relevant receptors in the rat bladder.

Hachi-mi-jio-gan extract (Harncare; HE), a galenical produced from the traditional Chinese herbal mixture Ba-Wei-Die-Huang-Wan, has been reported to improve lower urinary tract symptoms (LUTS) in patients. The present study was undertaken to clarify the pharmacological effects of HE on smooth muscle contraction and on pharmacologically relevant (muscarinic, 1,4-DHP and purinergic) receptors in the rat bladder. Additionally, the effects of repeated oral treatment with HE on the hepatic cytochrome P-450 (CYP) and on blood biochemical values in rats were examined. HE (10 mg/ml) inhibited significantly the acetylcholine-induced contraction of isolated rat bladder strips. The pD(2) value in the absence and presence of HE (10 mg/ml) was 5.14+/-0.16 and 3.99+/-0.17, respectively. HE (0.1 to 10 mg/ml) inhibited the specific binding of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS), (+)-[(3)H]PN 200-110 and alphabeta-methylene ATP [2,8-(3)H]tetrasodium salt ([(3)H]alphabeta-MeATP) in the rat bladder in a concentration-dependent manner. The respective IC(50) values were 6.85+/-0.94, 7.08+/-0.72 and 1.34+/-0.23 mg/ml. Based on IC(50) values, the binding activity of HE for purinergic receptors was shown to be significantly (about 7 times) greater than that for muscarinic and 1,4-DHP receptors. Repeated oral administration of HE (10, 30 and 100 mg/kg/day) for 4 weeks had little or no effect on the level and activity of hepatic CYP or on blood biochemical values in rats. In conclusion, the present study has shown that HE exerts significant binding activity for pharmacologically relevant receptors in the rat bladder and a relaxant effect on the acetylcholine-induced contraction of isolated muscle strips. HE seemed to exhibit little pharmacokinetic interaction with drugs.

The effects of erythrocyte deformability upon hematocrit assessed by the conductance method.

A comparative study of centrifugation and conductance methods for the estimation of cell volume fraction (phi) was performed to examine whether the strong forces exerted upon erythrocytes during centrifugation affect their volume, and the results are discussed in terms of erythrocyte deformability. Rabbit erythrocytes of four shapes (spherocytes, echinocytes, stomatocyte-like enlarged erythrocytes and discocytes) were prepared by controlling the pH of the suspending media. The packed cell volumes of the suspensions were measured by standard hematocrit determination methods using centrifugation in capillary tubes. Simultaneously, the same suspensions and their supernatants were used in dielectric spectroscopy measurements, and the low-frequency limits of their conductivities were used for the numerical estimation of phi. The hematocrit values of spherocytes and echinocytes were markedly less than the volume fractions obtained by the conductance method. Namely, the centrifugation reduced the cell volume. For enlarged erythrocytes and discocytes, however, the reduction of cell volume was not observed. These findings showed that phi obtained by the centrifugation method can be greatly affected by the deformability of the cells, but the level of the effect depends on the cell types. Consequently, phi obtained by the centrifugation method should be carefully interpreted.

Dielectric cytometry with three-dimensional cellular modeling.

We have developed what we believe is an efficient method to determine the electric parameters (the specific membrane capacitance C(m) and the cytoplasm conductivity kappa(i)) of cells from their dielectric dispersion. First, a limited number of dispersion curves are numerically calculated for a three-dimensional cell model by changing C(m) and kappa(i), and their amplitudes Deltaepsilon and relaxation times tau are determined by assuming a Cole-Cole function. Second, regression formulas are obtained from the values of Deltaepsilon and tau and then used for the determination of C(m) and kappa(i) from the experimental Deltaepsilon and tau. This method was applied to the dielectric dispersion measured for rabbit erythrocytes (discocytes and echinocytes) and human erythrocytes (normocytes), and provided reasonable C(m) and kappa(i) of the erythrocytes and excellent agreement between the theoretical and experimental dispersion curves.

Structure and conductance of aromatic and aliphatic dithioacetamide monolayers on Au(111).

The structure and electrical properties of self-assembled monolayers of cyclic aromatic and aliphatic dithioacetamides (1,4-bis(mercaptoacetamido)benzene and 1,4-bis(mercaptoacetamido)cyclohexane) and of mixed dithioacetamide/alkanethiol monolayers are characterized by X-ray photoelectron spectroscopy (XPS), scanning tunneling microscopy (STM) and contact angle goniometry. Both dithioacetamides are found to pack densely on Au(111), however the monolayers are poorly ordered as a result of hydrogen bond formation between the amide groups. The coassembly and the insertion method are compared for the formation of mixed dithioacetamide/alkanethiol monolayers. By coassembly, islands of dithioacetamides in a dodecanethiol matrix can only be obtained at a low dithioacetamide/dodecanethiol concentration ratio in solution (1/10) and by thermal annealing of the resulting monolayers. Small and well defined dithioacetamide domains are realized by insertion of dithioacetamides into defect sites of closely packed octanethiol monolayers. These domains are used to determine the molecular conductance by means of STM height profiles and molecular lengths resulting from density functional theory (DFT) calculations. The difference in the tunneling decay constant beta measured for aromatic dithioacetamides (beta = 0.74-0.76/A) and for aliphatic dithioacetamides (beta = 0.84-0.91/A) highlights the influence of the conjugation within the cyclic core on molecular conductance.

Dielectric inspection of erythrocyte morphology.

We performed a systematic study of the sensitivity of dielectric spectroscopy to erythrocyte morphology. Namely, rabbit erythrocytes of four different shapes were prepared by precisely controlling the pH of the suspending medium, and their complex permittivities over the frequency range from 0.1 to 110 MHz were measured and analyzed. Their quantitative analysis shows that the characteristic frequency and the broadening parameter of the dielectric relaxation of interfacial polarization are highly specific to the erythrocyte shape, while they are insensitive to the cell volume fraction. Therefore, these two dielectric parameters can be used to differentiate erythrocytes of different shapes, if dielectric spectroscopy is applied to flow-cytometric inspection of single blood cells. In addition, we revealed the applicability and limitations of the analytical theory of interfacial polarization to explain the experimental permittivities of non-spherical erythrocytes.

Microcrystalline composite particles of carbon nanotubes and calcium carbonate.

Coprecipitation of urea-melt modified carbon nanotubes and calcium carbonate from an aqueous solution by two methods yielded microcrystalline composite particles. Powders obtained by colloidal crystallization from a supersaturated solution that were isolated and dried soon after precipitation were a mixture of raspberry-shaped and rhombohedral particles. These were shown by infrared and X-ray diffraction analyses to be mainly calcite. Particles that were kept wet for 1 day or longer before being isolated were typically entirely rhombohedral with edge lengths in the range of 5-30 microm. Scanning electron microscopy investigations revealed that the nanotubes were adsorbed on the particle surface and also incorporated into the interior matrix. Removal of the calcium carbonate component by treating the particles with acid yielded nanotube shells whose size and shape reflected those of the original particles.

Temporal variation of dielectric properties of preserved blood.

Rabbit blood was preserved at 277 K in Alsever's solution for 37 days, and its dielectric permittivity was monitored in a frequency range from 0.05 to 110 MHz throughout the period. The relaxation time and Cole-Cole parameter of the interfacial polarization process for erythrocytes remained nearly constant during the first 20 days and then started to increase and decrease, respectively. On the other hand, the relaxation strength and the cell volume fraction continued to decrease for 37 days, but the decrease rates of both changed discontinuously on about the 20th day. Microscope observation showed that approximately 90% of the erythrocytes were spinous echinocytes at the beginning of preservation and started to be transformed into microspherocytes around the 20th day. Therefore, dielectric spectroscopy is a sensitive tool to monitor the deterioration of preserved blood accompanied by morphological transition of erythrocytes through the temporal variation of their dielectric properties.

A direct repeat of E-box-like elements is required for cell-autonomous circadian rhythm of clock genes.

BACKGROUND: The circadian expression of the mammalian clock genes is based on transcriptional feedback loops. Two basic helix-loop-helix (bHLH) PAS (for Period-Arnt-Sim) domain-containing transcriptional activators, CLOCK and BMAL1, are known to regulate gene expression by interacting with a promoter element termed the E-box (CACGTG). The non-canonical E-boxes or E-box-like sequences have also been reported to be necessary for circadian oscillation. RESULTS: We report a new cis-element required for cell-autonomous circadian transcription of clock genes. This new element consists of a canonical E-box or a non-canonical E-box and an E-box-like sequence in tandem with the latter with a short interval, 6 base pairs, between them. We demonstrate that both E-box or E-box-like sequences are needed to generate cell-autonomous oscillation. We also verify that the spacing nucleotides with constant length between these 2 E-elements are crucial for robust oscillation. Furthermore, by in silico analysis we conclude that several clock and clock-controlled genes possess a direct repeat of the E-box-like elements in their promoter region. CONCLUSION: We propose a novel possible mechanism regulated by double E-box-like elements, not to a single E-box, for circadian transcriptional oscillation. The direct repeat of the E-box-like elements identified in this study is the minimal required element for the generation of cell-autonomous transcriptional oscillation of clock and clock-controlled genes.

Comparative study of urea and betaine solutions by dielectric spectroscopy: liquid structures of a protein denaturant and stabilizer.

We performed dielectric spectroscopy measurements on aqueous solutions of glycine betaine (N,N,N-trimethylglycine), which is known to be a strong stabilizer of globular proteins, over a wide concentration range (3-62 wt %) and compared the results with our previously published data for aqueous solutions of urea, a representative protein denaturant. The hydration number of betaine (9), calculated on the basis of the reduction in the dielectric relaxation strength of bulk water with addition of betaine, is significantly larger than that of urea (2). Furthermore, the dielectric relaxation time increased with betaine concentration, while that remained nearly constant for the urea-water system over a wide concentration range. This difference between urea and betaine is probably related to their opposite effects on the protein stabilization.