Heart Rate Change While Drowsy Driving.

Heart rate (HR) change during sleepy driving has never been investigated. Healthy volunteers who planned to drive a long distance were recruited and monitored with a 24-hour Holter. Six healthy volunteers were enrolled. Their mean driving time was 297.7 ± 111 minutes. Mean duration of sleepy time while driving was 27 ± 24.5 minutes. Driving HR showed a trend for increment as compared to day time mean HR, from 85 ± 5.6 to 89.8 ± 5.6 beats/min (by 7%) (P = 0.093). Mean HR while sleepy driving significantly decreased to 81.5 ± 9.2 beats/min by 9.3% ± 7.4% (P = 0.046). This pilot study for the first time demonstrated that HR decreased while sleepy driving.

Synthesis, optical properties and preliminary in vitro photodynamic effect of pyridyl and quinoxalyl substituted chlorins.

A series of chlorophyll a-based chlorins conjugated with pyridyl or quinoxalyl group at different positions were synthesized, characterized and evaluated for their photodynamic effect in vitro. It was found that all the pyridyl and quinoxalyl chlorins showed promising photocytotoxicities but nontoxic without irradiation in HeLa cells, and the substituted types and positions had a significant influence on the photocytotoxicities of the chlorophyll a-based chlorins. All the chlorins with a pyridyl group at the C-D ring end exhibited relatively high photocytotoxicity as compared to those with 3(2)-pyridyl. Among them, compound 12 conjugated with a pyridyl group at its C12 position showed the best photodynamic effect in HeLa cells with an IC50 value of 0.033µM. These facts, associated with the relative high long wavelength absorptions of those chlorins may provide valuable ways to design and prepare promising photosensitizers for application in photodynamic therapy.

Cytotoxic cytochalasins from the endozoic fungus Phoma sp. of the giant jellyfish Nemopilema nomurai.

Four new cytochalasin derivatives (1-4), together with cytochalasin B (5), were isolated from the fungus Phoma sp. obtained from the giant jellyfish Nemopilema nomurai. The planar structure and relative stereochemistry were established by analysis of 1D and 2D NMR data. The absolute configuration was defined by the modified Mosher's method. The compounds showed significant cytotoxicity against a small panel of human solid tumor cell lines (A549, SK-OV-3, SK-MEL-2, XF 498, and HCT15) with IC(50) values in the range of 0.5-30 µM. The cytochalasin B (5) showed obvious cytotoxicity with IC(50) of 7.9 µM against HeLa human cervical carcinoma cells.

Alpha-helix 1 in the DNA-binding domain of heat shock factor 1 regulates its heat-induced trimerization and DNA-binding.

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. The role of alpha-helix1 (H1) in its DNA-binding domain (DBD) during HSF1 activation remains unknown. Here, HSF1 lacking H1 loses its heat-induced activity, suggesting the importance of the latter. Furthermore, the CD spectra and AMBER prediction show that this H1 deficiency does not change the structure of HSF1 monomer, but does impact its heat-induced trimerization. Point mutation showed that Phe18 in H1 interacts with Tyr60, and that Trp23 interacts with Phe104 by an aromatic-aromatic interaction. Thus, the presence of H1 stabilizes the DBD structure, which facilitates the heat-induced trimerization and DNA-binding of HSF1.

Environmental estrogenic effects and gonadal development in wild goldfish (Carassius auratus).

Serum vitellogenin (VTG) contents of wild goldfish (Carassius auratus) were investigated as a sensitive biomarker for artificial estrogenic compounds in aquatic environments. Goldfish was sampled from a pristine area, a river situated 5 km downstream from a sewage treatment works (STW), and also from the Young-San River in Korea. The female yolk precursor protein VTG was not detected when gonadosomatic index (GSI) was less than 0.85%, while VTG levels of >10 microg/ml were found in males whose GSI was less than 1.53%. In male goldfish sampled from STW and the Young-San River, the higher VTG corresponded to lower GSI. This study suggested a trend that gonad development was connected to VTG levels in both sexes, and the application of GSI and histological analysis provide an attractive possibility that it could be included in the panel of markers used for estrogenic activity investigation of aquatic environments.

Quadrature Frequency-Group Radar and its center estimation algorithms for small Vibrational Displacement.

The quadrature continuous-wave (QCW) radar has been extensively studied for small vibrational displacement detection such as non-contact sensing of human vital signals. One of the challenges of the QCW radar is the IQ-imbalance and DC-offset estimation by using curve fitting algorithms. Many algorithms have been proposed and have shown that the fitting error increases when the displacement length is small, in which case sufficient data is not provided to the algorithms. This paper presents a quadrature frequency-group (QFG) radar which utilizes a group of frequencies to enhance the fitting performance even with the small displacement. The grouped-frequencies in the QFG radar gives more data than the single-tone of the QCW radar under the same displacement condition. This paper presents the framework and properties of the QFG radar. Some fitting algorithms for the QFG radar are presented and the most adequate algorithm is suggested by simulation and experiments. Simulation and experimental results shows that the QFG radar outperforms the QCW radar. Specifically, it is shown that the fitting accuracy of the QFG radar is up to 100 times better than the QCW radar in the experiment.

Two distinct disulfide bonds formed in human heat shock transcription factor 1 act in opposition to regulate its DNA binding activity.

Under circumstances of heat stress, heat shock transcription factor 1 (HSF1) plays important roles in heat shock protein expression. In this study, an increasing concentration of dithiothreitol (DTT) was found to either enhance or inhibit the heat-induced trimerization of HSF1, suggesting the involvement of dual redox-dependent HSF1 activation mechanisms. Our in vitro experiments show that the heat-induced bonding between the cysteine C36 and C103 residues of HSF1 forms an intermolecular disulfide covalent bond (SS-I bond) and that it directly causes HSF1 to trimerize and bond to DNA. Gel filtration assays show that HSF1 can form intermolecular hydrophobic interaction-mediated (iHI-m) noncovalent oligomers. However, the lack of a trimerization domain prevents HSF1 activation, which suggests that iHI-m noncovalent trimerization is a precondition of SS-I bond formation. On the other hand, intramolecular SS-II bond (in which the C153, C373, and C378 residues of HSF1 participate) formation inhibits this iHI-m trimerization, thereby preventing SS-I bond formation and DNA binding. Thus, HSF1 activation is regulated positively by intermolecular SS-I bond formation and negatively by intramolecular SS-II bond formation. Importantly, these two SS bonds confer different DTT sensitivities (the SS-II bond is more sensitive). Therefore, a low concentration of DTT cleaves the SS-II bond but not the SS-I bond and thus improves DNA binding of HSF1, whereas a high concentration DTT cuts both SS bonds and inhibits HSF1 activation. We propose that these interesting effects further explain cellular HSF1 trimerization, DNA binding, and transcription when cells are under stress.

Apoptotic activity of fatty acid derivatives may correlate with their inhibition of DNA replication.

The apoptogenic and DNA damaging effects of (E)-10-oxooctadec-8-enoic acid (S5C) and (E)-9-oxooctadec-10-enoic acid (S6C), two structurally related fatty acids isolated from Red Alga Gracilaria verrucosa, were compared and their apoptosis-inducing properties characterized against human lung carcinoma (A549) cells. Significantly, the two acids decreased the rates of proliferation and viability (IC50 of approximately 170 and approximately 140 microM) as well as evidence of the induction of apoptosis. Cell morphological changes observed under light microscopy confirmed apoptosis occurrence. The results from Annexin V/PI dual staining and the cell cycle arrest assay indicated that S5C and S6C induced an earlier apoptosis of A549 cells in a concentration-dependent manner. We found that they induced DNA damage and inhibited DNA replication followed by S-phase arrest. In addition, the very sensitive alkaline micro-gel electrophoresis technique (comet assay) was used to estimate the compound-induced DNA single- and double-strand breaks. These findings suggest that S5C and S6C induced A549 cell apoptosis and their effects are associated with DNA damage. Therefore, S5C and S6C have the potential to be developed into anticancer agents due to their relatively easy synthesis and structural manipulation.

The possible roles for polyamines in the initiation process of SV40 DNA replication in vitro.

The polyamines are aliphatic cations which are present in millimolar concentrations in all mammalian cells, and are required for optimal growth of almost all cell types. In this study, the roles of polyamines in DNA replication in vitro and the mechanism by which polyamines affected DNA replication were examined using simian virus 40 DNA replication system in vitro. We found that polyamines inhibited DNA replication, but it is not clear at which stage this occurs. Spermidine inhibited the DNA cleavage by topoisomerase I at 8.0 mM, but stimulated its activity at 1.0 mM. Spermine also inhibited its activity at 4.0 mM, but stimulated at 1.0 mM. The ssDNA binding activity of replication protein A was slightly affected by polyamines. Polyamines, especially spermine, also significantly reduced polymerase alpha-primase activity at 133 microM. Taken together, we suggest that the major inhibition of SV40 DNA replication may be due to the inhibition of pol alpha-primase activity, and possible roles for polyamines in the initiation process are discussed.

Cytotoxicity of p-tyrosol and its derivatives may correlate with the inhibition of DNA replication initiation.

p-Tyrosol is a phenolic compound present in different dietary sources that can exert mild antioxidant properties based on in vitro and in vivo studies. In our study, two p-tyrosol derivatives (p-tyrosyl gallate and p-tyrosyl acetate) were synthesized and compared together with p-tyrosol and gallic acid for their cytotoxic activities on human cancer cells. p-Tyrosyl gallate had the most potent cytotoxicity and the major cytotoxic mechanism of its action was studied. We found that in HeLa cells, p-tyrosyl gallate can effectively induce cell cycle arrest during S phase and inhibited in vitro simian virus (SV40 DNA) replication. In addition, p-tyrosyl gallate can inhibit three important functional replication proteins (topoisomerase I, RPA and pol alpha-primase), especially pol alpha-primase. These results suggest that p-tyrosyl gallate-induced cell cycle arrest during S phase correlates with the inhibition of DNA replication. Pol alpha-primase may be the main target molecule. Taken together, we suggest that p-tyrosyl gallate is a strong anticancer drug candidate that warrants further investigation.

Anticancer effects of a novel chroman analog in HeLa cells are associated with G2­phase arrest and mitochondrial­mediated apoptosis.

In the present study, the anticancer activity of 1­[(3S,4R)­2,2­dimethyl­3­oxo­4­(2­piperidonyl)chroman­6­yl]­3­phenylurea (S32) was investigated by testing its effect in vitro on the growth of HeLa cells. First, we showed that the IC50 value of S32 was ~70 µM by using WST­8 assay, and that it significantly inhibited the proliferation and viability of HeLa cells in a dose­dependent manner after 48 h. Morphological changes in apoptotic cells included cellular shrinkage and nuclear condensation. The results of [3H]­thymidine incorporation and flow cytometric analysis indicated that S32 induced inhibition of DNA replication and G2­phase cell cycle arrest. Moreover, S32 induced the levels of reactive oxygen species (ROS) and decreased the mitochondrial membrane potential (MMP) in a time­dependent manner. Using Annexin V­FITC/propidium iodide (PI) dual staining assay, we found that S32 noticeably increased early apoptosis in HeLa cells in a time­dependent manner. The result of western blot analysis showed that the apoptotic induction was associated with an increase in Bax levels and a decrease in Bcl­2 levels, which led to activation of caspase­8, ­9 and ­3. Taken together, our findings demonstrated that S32 induces mitochondrial­mediated apoptosis in HeLa cells and suggest that S32 has potential as an anticancer drug.

3-Chloro-2,5-dihydroxybenzyl alcohol activates human cervical carcinoma HeLa cell apoptosis by inducing DNA damage.

We have isolated 3-chloro-2,5-dihydroxybenzyl alcohol (CHBA) from marine-derived fungus Aspergillus sp. and characterized its apoptosis-inducing properties against human cervical carcinoma (HeLa) cells. Significantly decreased rates of proliferation and viability (IC50 approximately 35 microM) as well as evidence of apoptosis were observed with CHBA. Nuclear changes observed under fluorescence microscopy confirmed apoptosis occurrence and showed a typical pattern of chromatin condensation. Furthermore, results from Annexin V-FITC/PI dual staining indicated that CHBA induced earlier apoptosis of HeLa cells in a concentration- and time-dependent manner. CHBA also induced cytochrome c release from mitochondria into the cytosol and subsequent caspase activation involving caspase-9 and -3 by Western blotting assay was observed. We also found that CHBA was able to induce DNA damage and inhibit DNA replication followed by S phase arrest. The very sensitive alkaline microgel electrophoresis technique (comet assay) was used for estimation of the CHBA-induced DNA single strand breaks. These findings suggest that CHBA induces HeLa cell apoptosis by cytochrome c release and caspase activation pathway and that the effect of CHBA on apoptosis of HeLa cells is associated with DNA damage. Because of the ease of synthesis and structural manipulation, CHBA may have the potential to be developed into an anticancer agent.

A sensitive and reliable quantification method for Bisphenol A based on modified competitive ELISA method.

Bisphenol A (BPA), generally known as bisphenols, has been identified as a potential estrogenic substance. BPA must be conjugated to carrier protein and BSA was commonly used. 4,4-Bis(4-hydroxyphenyl) valeric acid (BHPVA) has a bisphenolic structure and a long carbon chain with a reactive carboxyl group on the end. In this study, BHPVA-BSA was used to produce polyclonal antibody against bisphenolic structure, and a modified competitive ELISA method for quantification of BPA was developed. This system was based on BHPVA-BSA for polyclonal antibody production against bisphenolic structure, and BHPVA-HRP for determination of BPA substituting detection antibody in competitive reaction. Recovery was assessed at 10 different concentrations (2-1000 ng/ml) of BHPVA, and the recovery range was from 96.3% to 107.2%. The variation was from 6.2% to 9.8% for intra assay and from 10.1% to 12.6% for inter assay. The quadratic was used to establish the curve regression. The range was found to be between 2 and 1000 ng/ml. This modified competitive ELISA method has proven to be a very useful tool for quantification of BPA without the unexpected interaction of BSA and anti-BSA polyclonal antibody.

Endoscopic removal of a dental implant through a middle meatal antrostomy.

Ear nose and throat surgeons use endoscopic operations on the sinuses not only for chronic paranasal sinusitis but also for other operations. We report the case of a 52-year-old woman in whom we used an endoscopic technique to remove a dental implant that had been displaced into the maxillary sinus. We approached the sinus through the natural ostium, and the foreign body was removed through the widened ostium.

Mechanism of cell cycle arrest by (8E,13Z,20Z)-strobilinin/(7E,13Z,20Z)-felixinin from a marine sponge Psammocinia sp.

Furanosesterterpenes, isolated from a marine sponge Psammocinia sp. have been reported to display significant cytotoxicity to some cancer cell lines. In this study, EZZ, an inseparable 1:1 mixture of (8E,3Z,20Z)-strobilinin and (7E,3Z,20Z)-felixinin, showed significant antiproliferative effect on human cervix carcinoma cell line (HeLa). Cell cycle analysis revealed that EZZ could arrest HeLa cells in S phase with a concomitant decrease in the cell population of G1 phase. By using simian virus (SV40) DNA in vitro replication system, we found that EZZ could inhibit DNA replication, which suggests that EZZ-induced S phase arrest might be the direct result of blocked DNA synthesis. Furthermore, low concentration of EZZ was found to be capable of significantly inhibiting the DNA cleavage by topoisomerase I (topo I) and reducing the polymerase alpha-primase (pol alpha-primase) activity, while the ssDNA binding activity of replication protein A (RPA) was less affected. Taken together, these results suggest that EZZ-induced cell cycle arrest in S phase correlate with the inhibition of DNA replication, and topo I and pol alpha-primase might be the two main target molecules.

A novel bispidinone analog induces S­phase cell cycle arrest and apoptosis in HeLa human cervical carcinoma cells.

2,4,6,8-(3)-Tetranitrophenyl-3,7-diazabicyclo[3.3.1]nonan-9-one (B16), a bispidinone analog, was synthesized to investigate its effects on cell viability, the cell cycle, and apoptotic pathways in HeLa human cervical cancer cells. B16 decreased the percentage of viable cells in WST-8 assays, and morphological changes associated with apoptotic cell death were observed, including cell shrinkage and disruption. Annexin V-FITC/PI dual staining assays showed that B16 significantly increased the early apoptosis of HeLa cells after 24 h of treatment. Moreover, DNA content analysis and [3H]-thymidine incorporation assays showed that B16 induced S-phase cell cycle arrest and inhibited DNA replication after 24 h of treatment. Following treatment with 25 µM of B16, an increase in reactive oxygen species and a decrease in mitochondrial membrane potential were observed by flow cytometry. In addition, the expression levels of caspase cascade and Bcl-2 family proteins determined by western blotting suggested that the induction of apoptosis by B16 was associated with a caspase- and mitochondrial-dependent pathway in HeLa cells. In conclusion, B16 induced early apoptosis and S-phase cell cycle arrest in HeLa cells via a caspase- and mitochondrial­dependent pathway.

Polyacetylenes from a marine sponge Petrosia sp. inhibit DNA replication at the level of initiation.

In the course of our search for bioactive metabolites from the marine sponges collected from Korean water, we found that the polyacetylenes of marine sponge, genus Petrosia, deliver significant selective cytotoxicity against several human tumor cell lines. The effects of polyacetylene on DNA replication were examined using simian virus 40 DNA replication system in vitro. We found that polyacetylenes inhibited DNA replication, and predominantly inhibited the initiation stage of DNA replication. Polyacetylenes inhibited the DNA cleavage by topoisomerase I, and also significantly reduced polymerase alpha-primase activity. The ssDNA binding activity of replication protein A was little affected by polyacetylenes. We suggest that polyacetylenes might inhibit proteins required to establish replication forks during the initiation reaction, and their cytotoxicities might be related to the inhibitory effect they have on this fundamental cellular process.

Cytotoxicity of psammaplin A from a two-sponge association may correlate with the inhibition of DNA replication.

BACKGROUND: SV40 DNA replication system is a very useful tool to understand the mechanism of replication, which is a tightly regulated process. Many environmental and cellular factors can induce cell cycle arrest or apoptosis by inhibiting DNA replication. In the course of our search for bioactive metabolites from the marine sponges, psammaplin A was found to have some anticancer properties, the possible mechanism of which was studied. METHODS: Cell viability was determined by Cell Counting Kit-8 (CCK-8) to count living RAW264.7 cells by combining 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) and 1-methoxy-phenazine methosulfate (1-methoxy-PMS). The effect of psammaplin A on DNA replication was carried out in SV40 DNA replication system in vitro. The activities of topoisomerase I and polymerase alpha-primase were measured by the relaxation of superhelical plasmid DNA and the incorporation of [3H]dTTP to the template respectively. The ssDNA binding activity of RPA was assessed by Gel Mobility Shift Assay (GMSA). RESULTS: We have found that psammaplin A delivers significant cytotoxic activity against the RAW264.7 cell line. It was also found that psammaplin A could substantially inhibit SV40 DNA replication in vitro, in which polymerase alpha-primase is one of its main targets. CONCLUSION: Taken together, we suggest that psammaplin A-induced cytotoxicity may correlate with its inhibition on DNA replication. Psammaplin A has the potential to be developed as an anticancer drug.

Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene.

In the present study, differential gene expression in the uteri of ovariectomised (OVX) and pro-oestrous rats (OVX v. pro-oestrus pair) was investigated using cDNA expression array analysis. Differential uterine gene expression in OVX rats and progesterone (P(4))-injected OVX rats (OVX v. OVX + P(4) pair) was also examined. The uterine gene expression profiles of these two sets of animals were also compared for the effects of P(4) treatment. RNA samples were extracted from uterine tissues and reverse transcribed in the presence of [alpha(32)P]-dATP. Membrane sets of rat arrays were hybridised with cDNA probe sets. Northern blot analysis was used to validate the relative gene expression patterns obtained from the cDNA array. Of the 1176 cDNAs examined, 23 genes showed significant (>two-fold) changes in expression in the OVX v. pro-oestrus pair. Twenty of these genes were upregulated during pro-oestrus compared with their expression in the OVX rat uterus. In the OVX v. OVX + P(4) pair, 22 genes showed significant (>two-fold) changes in gene expression. Twenty of these genes were upregulated in the OVX + P(4) animals. The genes for nuclear factor I-XI, afadin, neuroligin 2, semaphorin Z, calpain 4, cyclase-associated protein homologue, thymosin beta-4X and p8 were significantly upregulated in the uteri of the pro-oestrus and OVX + P(4) rats of both experimental pairs compared with the OVX rat uteri. These genes appear to be under the control of P(4). One of the most interesting findings of the present study is the unexpected and marked expression of the neuroligin 2 gene in the rat uterus. This gene is expressed at high levels in the central nervous system and acts as a nerve cell adhesion factor. According to Northern blot analysis, neuroligin 2 gene expression was higher during the pro-oestrus and metoestrus stages than during the oestrus and dioestrus stages of the oestrous cycle. In addition, neuroligin 2 mRNA levels were increased by both 17beta-oestradiol (E(2)) and P(4), although P(4) administration upregulated gene expression to a greater extent than injection of E(2). These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E(2) and P(4), which are secreted periodically during the oestrous cycle.

Effect of a bispidinone analog on mitochondria­mediated apoptosis in HeLa cells.

The present study was carried out to investigate the effect of 2,4,6,8-tetraaryl-3,7-diazabicyclo[3.3.1]nonan-9-one (bispidinone) analogs on the in vitro growth of human cervical carcinoma (HeLa) cells. A series of 11 bispidinone analogs was synthesized with substituents, e.g., fluoro/methyl/ethyl/isopropyl/thiomethyl/methoxy groups, at various positions. These compounds were synthesized to identify which substituent and position induced the strongest cytotoxic effect in cancer cells. Among these synthetics, analog 9, which contains methoxy groups, had the most significant cytotoxic effect on HeLa cells, and its IC50 value was less than 13 µM. A WST-8 assay also showed that analog 9 inhibited the proliferation of HeLa cells. By using DNA content analysis, we found that analog 9 induced sub-G1 and G1 phase arrest in a time-dependent manner. A [3H]-thymidine incorporation assay suggested that analog 9 inhibited DNA replication in HeLa cells. On performing light microscopy, morphological changes such as cellular shrinkage and disruption, which are apoptotic features, were observed in HeLa cells. Annexin V/propidium iodide double staining and rhodamine-123 staining showed that analog 9 induced apoptosis and disrupted the intracellular mitochondrial membrane potential in HeLa cells. The western blot analysis results suggested that analog 9 induced mitochondria-mediated apoptosis. In addition, we have shown that analog 9 may play a role in the Fas signaling apoptotic pathway.