Inhibition of Crm1-p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation.

Poly(ADP-ribose) polymerase 1 (PARP-1) and p53 are two key proteins in the DNA-damage response. Although PARP-1 is known to poly(ADP-ribosyl)ate p53, the role of this modification remains elusive. Here, we identify the major poly(ADP-ribosyl)ated sites of p53 by PARP-1 and find that PARP-1-mediated poly(ADP-ribosyl)ation blocks the interaction between p53 and the nuclear export receptor Crm1, resulting in nuclear accumulation of p53. These findings molecularly link PARP-1 and p53 in the DNA-damage response, providing the mechanism for how p53 accumulates in the nucleus in response to DNA damage. PARP-1 becomes super-activated by binding to damaged DNA, which in turn poly(ADP-ribosyl)ates p53. The nuclear export machinery is unable to target poly(ADP-ribosyl)ated p53, promoting accumulation of p53 in the nucleus where p53 exerts its transactivational function.

Evening circadian oscillator as the primary determinant of rhythmic motivation for Drosophila courtship behavior.

Circadian clocks of Drosophila melanogaster motivate males to court females at a specific time of day. However, clock neurons involved in courtship rhythms in the brain of Drosophila remain totally unknown. The circadian locomotor behavior of Drosophila is controlled by morning (M cells) and evening (E cells) cells in the brain, which regulate morning and evening activities, respectively. Here, we identified the brain clock neurons that are responsible for the circadian rhythms of the close-proximity (CP) behavior that reflects male courtship motivation. Interestingly, the ablation or functional molecular clock disruption of E cells caused arrhythmic CP behavior, but that of M cells resulted in sustained CP rhythms even in constant darkness. In addition, the ablation of some dorsal lateral neurons (LNd) of E cells using neuropeptide-F (NPF)-GAL4 did not impair CP rhythms. These findings suggested that the NPF-negative LNds and DN1s of E cells include cells essential for circadian CP behavior in Drosophila.

Drosophila melanogaster as a model for understanding polyADP-ribosylation.

PolyADP-ribosylation is a post-translational modification which is involved in various physiological processes including maintenance of genome stability through DNA repair, regulation of transcription, and development. This process is also involved in pathological events such as cell death. Here, we review the effect of polyADP-ribosylation in signal transduction pathways in Drosophila melanogaster system. It is hoped that such an insight paves the way to develop therapeutics for human diseases.

Reduction of human T-cell leukemia virus type-1 infection in mice lacking nuclear factor-kappaB-inducing kinase.

Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia and inflammatory disorders. Aberrant activation of nuclear factor-kappaB (NF-kappaB) has been linked to HTLV-1 pathogenesis and to various kinds of cancers, including adult T-cell leukemia. NF-kappaB-inducing kinase (NIK) is critical for non-canonical activation of NF-kappaB and for the development of lymphoid organs. HTLV-1 activates NF-kappaB by the non-canonical pathway, but examination of the role of NIK in proliferation of HTLV-1-infected cells in vivo has been hindered by lack of a suitable animal model. Alymphoplasia (aly/aly) mice bear a mutation of NIK, resulting in defects in the development of lymphoid organs and severe deficiencies in both humoral and cell-mediated immunity. In the present study we therefore used a mouse model of HTLV-1 infection with aly/aly mice. The number of HTLV-1-infected cells in the reservoir organs in aly/aly mice was significantly smaller than in the control group 1 month after infection. In addition, aly/aly mice did not maintain provirus for 1 year and antibodies against HTLV-1 were undetectable. These results demonstrate that the absence of functional NIK impairs primary HTLV-1 proliferation and abolishes the maintenance of provirus. Interestingly, clonal proliferation of HTLV-1-infected mouse cells was not detected in aly/aly mice, which is consistent with the lack of HTLV-1 persistence. These observations imply that the clonal proliferation of HTLV-1-infected cells in secondary lymphoid organs might be important for HTLV-1 persistence.

Differential circadian expression of endothelin-1 mRNA in the rat suprachiasmatic nucleus and peripheral tissues.

The vasoconstrictor endothelin-1 (ET-1) is implicated in normal neuronal functions. Here we show the circadian expression of ET-1 mRNA in the rat suprachiasmatic nucleus (SCN) that is considered to be the location of the central circadian pacemaker, as well as in peripheral tissues including the brain, heart, and lungs. The expression of ET-1 in the SCN oscillated with a peak at Zeitgeber time (ZT) 4 under light-dark conditions. A significant number of cells in the SCN was stained with ET-1 probe during circadian time (CT) 6, but there was no significant staining at CT18 by mRNA in situ hybridization. The circadian rhythm of ET-1 mRNA in the whole brain also oscillated, but peaked at ZT20. Endothelin-1 expression in the lungs and heart peaked at ZT12 and ZT20, respectively. The results are the first description of the circadian expression of ET-1 mRNA. The diversity of rhythmic expressions among the SCN, whole brain, lungs and heart suggests that ET-1 has different functions in these tissues.

Expression of human Gaucher disease gene GBA generates neurodevelopmental defects and ER stress in Drosophila eye.

Gaucher disease (GD) is the most common of the lysosomal storage disorders and is caused by defects in the GBA gene encoding glucocerebrosidase (GlcCerase). The accumulation of its substrate, glucocylceramide (GlcCer) is considered the main cause of GD. We found here that the expression of human mutated GlcCerase gene (hGBA) that is associated with neuronopathy in GD patients causes neurodevelopmental defects in Drosophila eyes. The data indicate that endoplasmic reticulum (ER) stress was elevated in Drosophila eye carrying mutated hGBAs by using of the ER stress markers dXBP1 and dBiP. We also found that Ambroxol, a potential pharmacological chaperone for mutated hGBAs, can alleviate the neuronopathic phenotype through reducing ER stress. We demonstrate a novel mechanism of neurodevelopmental defects mediated by ER stress through expression of mutants of human GBA gene in the eye of Drosophila.

Approaches to separate and identify polyADP-ribosylated proteins using poly(ADP-ribose) glycohydrolase-knockout Drosophila.

PolyADP-ribosylation plays an essential function in maintenance of genomic stability and cell survival. Although there are several proteins served as acceptor proteins in vitro, there are few proteins in vivo that are identified, including poly(ADP-ribose) polymerase-1. We have been studying to analyze the mechanism of neuronal cell death observed in poly(ADP-ribose) glycohydrolase (PARG)-knockout Drosophila melanogaster that shows accumulation of polyADP-ribosylated proteins in the brain. As the first step, we have been trying to isolate the polyADP-ribosylated accepter proteins from the PARG-knockout fly. The strategy is to extract the polyADP-ribosylated proteins and isolate them with affinity chromatography using monoclonal antibody against poly(ADP-ribose) (PAR) (10H). The bound fraction was eluted by buffer containing salt. Next, part of eluted fraction is treated with NaOH for separating the proteins from PAR chain. Nontreated fraction and treated fraction were separated with two-dimensional gel electrophoresis. After protein staining, the specific spots that were newly found after NaOH treatment were candidate acceptor proteins for polyADP-ribosylation in vivo and could be analyzed with liquid chromatography-mass spectrometry. We present the procedure to this approach.

Circadian clock in Ciona intestinalis revealed by microarray analysis and oxygen consumption.

The molecular mechanisms of the endogenous circadian clocks that allow most animals to adapt to environmental cycles have recently been uncovered. The draft genome of the ascidian, Ciona intestinalis, a model animal that is close to vertebrates, has been described. However, the C. intestinalis genome lacks the canonical clock genes such as Per, Bmal and Clock that are shared by vertebrates and insects. Here, we found the circadian rhythms at the physiological and molecular levels. The oxygen consumption rate was lower during the light phase and higher during the dark phase during a day, and the rhythm highly damped and continued under constant darkness. From the microarray analysis, the 396 spots (1.8% of the total; corresponding to 388 clones) were extracted as candidates for circadian expression. We confirmed the circadian expression of several candidate genes by northern blotting. Furthermore, three of four rhythmic expressed genes showed phase-shifts to prolonged light period. However, most of known clock genes did not oscillate. These data suggest that C. intestinalis have a unique molecular circadian clock and the daily environmental change is not such a strong effect for sea squirt in its evolution when compared to vertebrates and insects.

Entrainment of Drosophila circadian clock to green and yellow light by Rh1, Rh5, Rh6 and CRY.

Light is one of the most important time cues for entrainment of the circadian clock. Drosophila circadian photoreception is mediated by cryptochrome in clock neurons and by rhodopsins in photic organs. We generated Rh5 mutants to elucidate circadian photoreception by rhodopsins. The Rh1, Rh5 and Rh6 mutants were combined with cry, and entrained to a 6-h delayed photoperiod. The cry, Rh1, Rh5 and Rh6 quadruple mutant became entrained by white light. In contrast, reentrainment to green and yellow light was abolished in the cry, Rh1, Rh5 and Rh6 quadruple mutant, and remarkably slowed in the cry, Rh1 and Rh6 triple mutant. These results suggest that cry, Rh1, Rh5 and Rh6 are essential for circadian photoentrainment to green and yellow light.

Circadian entrainment to red light in Drosophila: requirement of Rhodopsin 1 and Rhodopsin 6.

Like most other insects, Drosophila visual system is sensitive to the spectrum of light from ultraviolet to green but is insensitive to red light. The circadian rhythms of Drosophila, however, can be entrained by red light through unknown mechanisms. Here, we discovered the corresponding organ and photopigments responsible for the circadian entrainment of Drosophila to red light from light emitting diode. The entrainment was eliminated by the loss of photic input from compound eyes using eya(2) or norpA(p24). We also found that the double Rhodopsin 1 (ninaE) and Rhodopsin mutant was not entrained to red light. These results indicate that Drosophila can entrain the circadian rhythm to red light through Rhodopsin 1 and Rhodopsin 6 in compound eyes.

Altered gene expression in Opisthorchis viverrini-associated cholangiocarcinoma in hamster model.

Cholangiocarcinoma (CCA) induced by liver fluke (Opisthorchis viverrini, Ov) infection is one of the most common and serious disease in northeast Thailand. To elucidate the molecular mechanism of cholangiocarcinogenesis induced by Ov infection, we employed a hamster model of CCA induced by Ov and N-nitrosodimethylamine and analyzed candidate genes involved in CCA using fluorescence differential display-PCR. Of 149 differentially amplified bands we identified, the upregulation of 23 transcripts and downregulation of 1 transcript related to CCA hamsters were confirmed by a reverse northern macroarray blot. The upregulated genes include signal transduction protein kinase A regulatory subunit Ialpha (Prkar1a), myristoylated alanine-rich protein kinase C substrate, transcriptional factor LIM-4-only domain, oxysterol-binding protein involved in lipid metabolism, splicing regulatory protein 9, ubiquitin conjugating enzyme involved in protein degradation, beta tubulin, beta actin, and collagen type VI. Quantitative real-time PCR confirmed that the expression of Prkar1a was significantly higher in CCA and its precursor lesion when compared with normal liver and normal gall bladder epithelia (P<0.05). Prkar1a expression tended to increase along with the progression of biliary transformation from hyperplasia and precancerous lesions to carcinoma. These findings contribute to our understanding of the processes involved in the molecular carcinogenesis of CCA in order to provide a unique perspective on the development of new chemotherapeutics in future.

Genetic and environmental determinants of risk for cholangiocarcinoma via Opisthorchis viverrini in a densely infested area in Nakhon Phanom, northeast Thailand.

Infection with Opisthorchis viverrini (OV) is associated with cholangiocarcinoma. OV is common in northeast Thailand, but less than 10% of the inhabitants develop cholangiocarcinoma. Animal experiments suggest that OV infection alone does not cause cholangiocarcinoma, and thus other environmental and genetic factors may play a role in causation. We conducted a population-based case-control study in which sex, age and place of residence were matched individually. Polymorphisms of GSTM1 and GSTT1 alone were not associated with risk for cholangiocarcinoma, while an elevated level of antibodies against OV (ELISA) > or = 0.200 was the strongest risk indicator (odds ratio as compared to that <0.200 = 27.09 [95% confidence interval (CI): 6.30-116.57]. Compared to subjects who had a normal antibody range and the wild-type GSTM1 gene, those who had elevated antibodies had higher odds ratios of 10.34 (95% CI: 1.31-81.63) [corrected] for wild-type GSTM1 and 18.00 (95% CI: 3.33-97.40) [corrected] for the null variant thereof, respectively. Past and current regular drinkers of alcohol had higher risk [odds ratio = 5.39 (95% CI: 1.11-26.06) and 4.82 (95% CI: 1.29-18.06), respectively]. Eating fermented products was an independent risk factor. Smokers or consumers of fermented fish had substantially increased risk if they were past or current drinkers. Infection with OV correlates strongly with cholangiocarcinoma, susceptibility to which may be possibly associated with GSTM1 polymorphism. Alcohol may affect metabolic pathways of endogenous and exogenous nitrosamines.

Effect of feeding on peripheral circadian rhythms and behaviour in mammals.

Although feeding time is a dominant cue for circadian rhythms in mammalian peripheral tissue, the effect of feeding and fasting on circadian gene expression and behaviour is unknown. Here we report that fasting does not affect the phase of rhythmic mRNA expression levels of the clock genes, mPer1, mPer2 and of the clock controlled gene, mDBP. However, the levels of each of these genes were significantly altered in different ways and recovered by feeding. We also found that feeding enhances phase-shifting to a new light-dark cycle of rhythmic mPer2 mRNA expression in the heart. Furthermore, feeding enhances the phase-shifting to new light-dark cycle of behaviour more than fasting. Our data indicate that feeding is an important cue for circadian behaviour rhythms as well as for the photo-entrainment of peripheral clock gene expression.

Loss of poly(ADP-ribose) glycohydrolase causes progressive neurodegeneration in Drosophila melanogaster.

Poly(ADP-ribosyl)ation has been suggested to be involved in regulation of DNA repair, transcription, centrosome duplication, and chromosome stability. However, the regulation of degradation of poly(ADP-ribose) and its significance are not well understood. Here we report a loss-of-function mutant Drosophila with regard to poly(ADP-ribose) glycohydrolase, a major hydrolyzing enzyme of poly(ADP-ribose). The mutant lacks the conserved catalytic domain of poly(ADP-ribose) glycohydrolase, and exhibits lethality in the larval stages at the normal development temperature of 25 degrees C. However, one-fourth of the mutants progress to the adult stage at 29 degrees C but showed progressive neurodegeneration with reduced locomotor activity and a short lifespan. In association with this, extensive accumulation of poly(ADP-ribose) could be detected in the central nervous system. These results suggest that poly(ADP-ribose) metabolism is required for maintenance of the normal function of neuronal cells. The phenotypes observed in the parg mutant might be useful to understand neurodegenerative conditions such as the Alzheimer's and Parkinson's diseases that are caused by abnormal accumulation of substances in nervous tissue.

The genetic background as a determinant of human T-cell leukemia virus type 1 proviral load.

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked with HTLV-1-associated diseases. HTLV-1 proviral load is higher in persons with adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis than in asymptomatic carriers. However there are little data available on the factors controlling HTLV-1 proviral load in carriers. To study the effect of genetic background on HTLV-1 proviral load, we employed a mouse model of HTLV-1 infection that we had established. Here we analyzed nine strains of mice and found there is a great variation of proviral load among mouse strains that is not necessarily dependent on major histocompatibility complex. The antibody response is also different among these strains. To our knowledge, this is the first demonstration of the importance of the genetic background other than major histocompatibility complex controlling the HTLV-1 proviral load.

Cell-free human T-cell leukemia virus type 1 binds to, and efficiently enters mouse cells.

Human T-cell leukemia virus type 1 (HTLV-1) is an etiologic agent of adult T-cell leukemia / lymphoma and other HTLV-1-associated diseases. However, the interaction between HTLV-1 and T cells in the pathogenesis of these diseases is poorly understood. Mouse cells have been reported to be resistant to cell-free HTLV-1 infection. However, we recently reported that HTLV-1 DNA could be observed 24 h after cell-free HTLV-1 infection of mouse cell lines. To understand HTLV-1 replication in these cells in detail, we concentrated the virus produced from c77 feline kidney cell line and established an efficient infection system. The amounts of adsorption of HTLV-1 are larger in mouse T cell lines, EL4 and RLm1, than those in human T cell lines, Molt4 and HUT78, and are similar to that in human kidney cell line, 293T. Unexpectedly, however, the amounts of entry of HTLV-1 are about 10-fold larger in the two mouse cell lines than those in the three human cell lines employed. Moreover, viral DNA was detectable from 1 h in EL4 and RLm1 cells, but only from 2 - 3 h in 293T, Molt4 and HUT78 cells. However, the amount of viral DNA in EL4 cells became smaller than that in Molt4 cells. HTLV-1 expression could be detected until day 1 - 2 in RLm1 and EL4 cells, and until day 4 in Molt4 cells. Our results suggest that mouse cell experiments would give useful information to dissect the early steps of cell-free HTLV-1 infection.

Overexpression of poly(ADP-ribose) polymerase disrupts organization of cytoskeletal F-actin and tissue polarity in Drosophila.

Poly(ADP-ribose) polymerase (PARP) may play important roles in nuclear events such as cell cycle, cell proliferation, and maintenance of chromosomal stability. However, the exact biological role played by PARP or how PARP is involved in these cellular functions is still unclear. To elucidate the biological functions of PARP in vivo, we have constructed transgenic flies that overexpress Drosophila PARP in the developing eye primordia. These flies showed mild roughening of the normally smooth ommatidial lattice and tissue polarity disruption caused by improper rotation and chirality of the ommatidia. To clarify how this phenotypical change was induced, here we analyzed transgenic flies overexpressing PARP in the developing eye, embryo, and adult in detail. PARP mRNA level and the phenotype were enhanced in flies carrying more copies of the transgene. Developing eyes from third instar larvae were analyzed by using the neural cell marker to examine the involvement of PARP in cell fate. Morphological disorder of non-neuronal accessory cells was observed in PARP transgenic flies. Interestingly, overexpression of PARP did not interfere with the cell cycle or apoptosis, but it did disrupt the organization of cytoskeletal F-actin, resulting in aberrant cell and tissue morphology. Furthermore, heat-induced PARP expression disrupted organization of cytoskeletal F-actin in embryos and tissue polarity in adult flies. Because these phenotypes closely resembled mutants or transgenic flies of the tissue polarity genes, genetic interaction of PARP with known tissue polarity genes was examined. Transgenic flies expressing either PARP or RhoA GTPase in the eye were crossed, and co-expression of PARP suppressed the effect of RhoA GTPase. Our results indicate that PARP may play a role in cytoskeletal or cytoplasmic events in developmental processes of Drosophila.

Subcellular localization of poly(ADP-ribose) glycohydrolase in mammalian cells.

Posttranslational modification plays important roles in a range of cellular functions. Poly(ADP-ribosyl)ation influences DNA repair, transcription, centrosome duplication, and chromosome stability. Poly(ADP-ribose) attached to acceptor proteins should be properly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). However the subcellular localization and the role of PARG have not been well characterized. Here, we transiently expressed GFP- or Myc-tagged human PARG in mammalian cells and revealed that the subcellular distribution of human PARG changes dramatically during the cell cycle. GFP-hPARG is found almost exclusively in the nucleus during interphase. During mitosis, most GFP-hPARG protein localizes to the cytoplasm and hardly any GFP-hPARG protein is found associated with the chromosomes. Furthermore, we found that GFP-hPARG localizes to the centrosomes during mitosis. Our findings suggest that shuttling of PARG between nucleus and cytoplasm and proper control of poly(ADP-ribose) metabolism throughout the cell cycle may play an important role in regulating cell cycle progression and centrosome duplication.

Integration of human T-cell leukemia virus type 1 in genes of leukemia cells of patients with adult T-cell leukemia.

Adult T-cell leukemia (ATL) occurs after a long latent period of persistent infection by human T-cell leukemia virus type 1 (HTLV-1). However, the mechanism of oncogenesis by HTLV-1 remains to be clarified. It was reported that the incidence curve of ATL versus age was consistent with a multistage carcinogenesis model. Although HTLV-1 is an oncogenic retrovirus, a mechanism of carcinogenesis in ATL by insertional mutagenesis as one step during multistage carcinogenesis has not been considered thus far, because the exact integration sites on the chromosome have not been analyzed. Here we determined the precise HTLV-1 integration sites on the human chromosome, by taking advantage of the recently available human genome database. We isolated 25 integration sites of HTLV-1 from 23 cases of ATL. Interestingly, 13 (52%) of the integration sites were within genes, a rate significantly higher than that expected in the case of random integration (P = 0.043, chi(2) test). These results suggest that preferential integration into genes at the first infection is a characteristic of HTLV-1. However considering that some of the genes are related to the regulation of cell growth, the integration of HTLV-1 into or near growth-related genes might contribute to the clonal selection of HTLV-1-infected cells during multistage carcinogenesis of ATL.