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.

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.

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.

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.

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.

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.

Knockdown of XAB2 enhances all-trans retinoic acid-induced cellular differentiation in all-trans retinoic acid-sensitive and -resistant cancer cells.

Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) is composed of 855 amino acids, contains 15 tetratricopeptide repeat motifs, and associates with Cockayne syndrome group A and B proteins and RNA polymerase II, as well as XPA. In vitro and in vivo studies showed that XAB2 is involved in pre-mRNA splicing, transcription, and transcription-coupled DNA repair, leading to preimplantation lethality, and is essential for mouse embryogenesis. Retinoids are effective for the treatment of preneoplastic diseases including xeroderma pigmentosum and other dermatologic diseases such as photoaging. We therefore focused on defining the effect of XAB2 on cellular differentiation in the presence of ATRA treatment. In the present study, we showed that overexpression of XAB2 inhibited ATRA-induced cellular differentiation in human rhabdomyosarcoma cell line, and that knockdown of XAB2 by small interfering RNA (siRNA) increased ATRA-sensitive cellular differentiation in the human promyelocytic leukemia cell line HL60 at both physiologic (10(-9)-10(-8) mol/L) and therapeutic (10(-7) mol/L) concentrations of ATRA. Moreover, we found that XAB2 was associated with retinoic acid receptor alpha (RARalpha) and histone deacetylase 3 in the nuclei. Finally, using siRNA against XAB2, we showed that the ATRA-resistant neuroblastoma cell line IMR-32 underwent cellular differentiation induced by ATRA at a therapeutic concentration (10(-6) mol/L). These results strongly suggest that XAB2 is a component of the RAR corepressor complex with an inhibitory effect on ATRA-induced cellular differentiation and that XAB2 plays a role in ATRA-mediated cellular differentiation as an important aspect of cancer therapy.

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.

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.

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.

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.

Liquid structure of the urea-water system studied by dielectric spectroscopy.

Dielectric spectroscopy measurements for aqueous urea solutions were performed at 298 K through a concentration range from 0.5 to 9.0 M with frequencies between 200 MHz and 40 GHz. Observed dielectric spectra were well represented by the superposition of two Debye type relaxation processes attributable to the bulk-water clusters and the urea-water coclusters. Our quantitative analysis of the spectra shows that the number of hydration water molecules is approximately two per urea molecule for the lower concentration region below 5.0 M, while the previous molecular dynamics studies predicted approximately six water molecules. It was also indicated by those studies, however, that there are two types of hydration water molecule in urea solution, which are strongly and weakly associated to the urea molecule, respectively. Only the strongly associated water was distinguishable in our analysis, while the weakly associated water exhibited the same dynamic feature as bulk water. This implies that urea retains the weakly associated water in the tetrahedral structure and, thus, is not a strong structure breaker of water. We also verified the model of liquid water where water consists of two states: the icelike-ordered and dense-disordered phases. Our dielectric data did not agree with the theoretical prediction based on the two-phase model. The present work supports the argument that urea molecules can easily replace near-neighbor water in the hydrogen-bonding network and do not require the presence of the disordered phase of water to dissolve into water.

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.

Dielectric dispersion of short single-stranded DNA in aqueous solutions with and without added salt.

Dielectric spectroscopy measurements were performed for aqueous solutions of short single-stranded DNA with 30 to 120 bases of thymine over a frequency range of 10;{5} to 10;{8}Hz . Dielectric dispersion was found to include two relaxation processes in the ranges from 10;{5} to 10;{6} and from 10;{6} to 10;{8}Hz , respectively, with the latter mainly discussed in this study. The dielectric increment and the relaxation time of the high-frequency relaxation of DNA in solutions without added salt exhibited concentration and polymer-length dependences eventually identical to those for dilute polyion solutions described in previous studies. For solutions with added salt, on the other hand, those dielectric parameters were independent of salt concentration up to a certain critical value and started to decrease with further increasing salt concentration. This critical behavior is well explained by our newly extended cell model that takes into account the spatial distribution of loosely bound counterions around DNA molecules as a function of salt concentration.

The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks.

BACKGROUND: Circadian rhythms are endogenous, self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes. The circadian organization of these processes in mammals is governed by the master oscillator within the suprachiasmatic nuclei (SCN) of the hypothalamus. Recent findings revealed that circadian oscillators exist in most organs, tissues, and even in immortalized cells, and that the oscillators in peripheral tissues are likely to be coordinated by SCN, the master oscillator. Some candidates for endogenous entrainment factors have sporadically been reported, however, their details remain mainly obscure. RESULTS: We developed the in vitro real-time oscillation monitoring system (IV-ROMS) by measuring the activity of luciferase coupled to the oscillatory gene promoter using photomultiplier tubes and applied this system to screen and identify factors able to influence circadian rhythmicity. Using this IV-ROMS as the primary screening of entrainment factors for circadian clocks, we identified 12 candidates as the potential entrainment factor in a total of 299 peptides and bioactive lipids. Among them, four candidates (endothelin-1, all-trans retinoic acid, 9-cis retinoic acid, and 13-cis retinoic acid) have already been reported as the entrainment factors in vivo and in vitro. We demonstrated that one of the novel candidates, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), a natural ligand of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), triggers the rhythmic expression of endogenous clock genes in NIH3T3 cells. Furthermore, we showed that 15d-PGJ2 transiently induces Cry1, Cry2, and Roralpha mRNA expressions and that 15d-PGJ2-induced entrainment signaling pathway is PPAR-gamma--and MAPKs (ERK, JNK, p38MAPK)-independent. CONCLUSION: Here, we identified 15d-PGJ2 as an entrainment factor in vitro. Using our developed IV-ROMS to screen 299 compounds, we found eight novel and four known molecules to be potential entrainment factors for circadian clocks, indicating that this assay system is a powerful and useful tool in initial screenings.

Dielectric dispersion for short double-strand DNA.

A complex dielectric constant for double-strand DNA molecules with a length of not greater than 120 base pairs in an aqueous solution containing 30 mM NaCl was systematically measured as a function of chain length in such a way that experimental uncertainties associated with the molecular-weight distribution of specimens were virtually excluded. In contrast to the past experimental and theoretical studies for much longer DNA molecules, both the molar specific dielectric increment and the relaxation time are proportional to the chain length. These scaling rules cannot be accounted for by any theory so far proposed that gives analytical expressions for those two quantities in the long-chain limit.

Switches from pi- to sigma-bonding complexes controlled by gate voltages.

A conjugated polymer/metal ion/liquid-crystal molecular system was set between source and drain electrodes with a 100 nm gap. When gate voltage (Vg) increases, the current between source and drain electrodes increases. Infrared spectra show this system to be composed of pi and sigma complexes. At Vg = 0, the pi complex dominates the sigma complex, whereas the sigma complex becomes dominant when Vg is switched on. Calculations found that the pi complex has lower conductivity than the sigma complex.

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.

Impact of micropatterned surfaces on neuronal polarity.

Experimental control over cellular polarity in a neuronal network is a promising tool to study synapse formation and network behavior. We aimed to exploit a mechanism described by Stenger et al. [J. Neurosci. Methods 82 (1998) 167] to manipulate the direction of axonal versus dendritic outgrowth on a micropattern. The group had used laser ablation to create patterns of aminated silanes for cell attachment on a background of repellent fluorinated silanes. The pattern offered continuous adhesive pathways for axonal and interrupted pathways for dendritic outgrowth. By microcontact printing, we created similar patterns containing continuous and interrupted pathways consisting of extracellular matrix proteins on a background of polystyrene. Neuronal polarity was determined on the functional level through double patch clamp measurements, detecting synapses and their orientation. Although our pattern reproduced the properties that were assumed to be critical for the described effect, namely contrasting pathways of different adhesiveness, we failed to reproduce the above results. It is indicated that other qualities of alternative pathways than mere differences in adhesiveness are required to orient neuronal polarity in vitro. We suggest that the effect observed by Stenger et al. has to be attributed to less universal characteristics of the micropattern, e.g. to the specific chemical groups that were utilized.