Abstract The aim of the present study was to evaluate whether bacterial vaginosis (BV) diagnosed in early pregnancy and treated with vaginal clindamycin affects pregnancy outcome, and to investigate the predictive value of interleukins-6 (IL-6) and -8 (IL-8), and insulin-like growth factor-binding protein-1 (IGFBP-1) in cervical secretions, separately and combined by cervical measurement with transvaginal ultrasonography, on preterm delivery. A further aim was to analyze retrospectively the significance of absent or reversed end-diastolic velocity (AREDV) in the umbilical artery on perinatal outcome, and to investigate the short- and long-term outcome of infants born prematurely as a result of various causes (indicated preterm birth, preterm premature rupture of the membranes=PPROM). Bacterial vaginosis (BV) was screened in 1956 women in a low-risk population at the first antenatal visit, using Gram stain. One hundred and one of 143 BV-positive women were randomized to receive vaginal clindamycin or placebo. Seventy-seven women at 22-32 gestational weeks with premature uterine contractions, and 78 controls were recruited for assay of cervical IL-6, IL-8-, and IGFBP-1, and ultrasonographic measurements, which were repeated twice at two-week intervals. Eighty-three women with AREDV in the umbilical artery in high-risk pregnancies at less than 34 gestational weeks (e.g. pre-eclampsia, small-for-gestational age [SGA]) between the years 1988-95 were analyzed retrospectively as regards perinatal outcome. Further, for 103 women between the 24th and the 33rd week of pregnancy, delivered by cesarean section because of maternal or fetal indications, and for 103 matched women, between the years 1990-97, their infants were analyzed as regards neonatal mortality and morbidity, and the outcome at one year of corrected age. Similarly, 78 women with PPROM at gestational weeks 17-30, and 78 controls were also analyzed. The prevalence of BV was 7.3% (143/1956) and the preterm birth rate in women with BV was 9.9%. Preterm birth occurred in 21% vs. 0% according to whether or not BV persisted. The preterm birth rate was 14% in the clindamycin group vs. 6% in the placebo group. Cervical IL-6 at a concentration of 128 ng/L had a 73% sensitivity and 77% specificity in predicting preterm birth (35% vs. 6%). The combination of IL-6 and a cervical index of > 0.2 increased the specificity to 97%, the sensitivity falling to 45%. Concentrations of IGFBP-1 were most elevated (> 21 μg/mL) in cases with neonatal infections (36% vs. 2%). In cases of absent end-diastolic velocity (AEDV) the perinatal mortality (PNM) rate was 9%, compared with 36% in the reversed end-diastolic velocity (REDV) group. Respiratory distress (RDS) and hypoglycemia, and chronic lung disease (CLD; 15% vs. 3%) occurred significantly more often in the indicated than in the spontaneously preterm infants. The PPROM infants had more limb contractures (8% vs. 0%) and pulmonary hypoplasia (12% vs. 5%) and more chronic lung problems up to one year of age than the spontaneously preterm born infants without PPROM. The persistence of pregnancy BV is a risk factor for preterm birth, but vaginal clindamycin used in a low-risk population in early pregnancy is of no use in reducing the preterm birth rate in cases of BV. The level of IL-6 has a relatively low sensitivity and a limited role as a single method in clinical decision making but in combination with cervical examination by ultrasonography it seems to have a predictive role in cases of threatened preterm birth. A finding of AREDV in the umbilical artery is a warning signal of threatened fetal asphyxia. Infants born after indicated preterm delivery (for fetal or maternal reasons) or PPROM are at risk of later chronic lung disease.

Previous publications from this laboratory demonstrated that administration of leukemia inhibitory factor (LIF) (125 µg/kg) to young, male Sprague-Dawley rats at 6, 24, and 48 h after middle cerebral artery occlusion (MCAO) reduced infract volume, improved sensimotor skills, and alleviated damage to white matter at 72 h after the injury. In vitro studies using cultured oligodendrocytes (OLs) showed that LIF (200 ng/ml) also protects against 24 h of oxygen-glucose deprivation through activation of Akt signaling and upregulation of the antioxidant enzymes peroxiredoxin IV and metallothionein III. Other groups have demonstrated that LIF reduces neurodegeneration in animal models of disease, but the neuroprotective mechanisms of LIF during permanent ischemia have not yet been examined. The overall hypothesis to be tested in this project is whether LIF exerts similar protective mechanisms against neurons during ischemia through increased antioxidant enzyme expression in neurons. In the first set of experiments, superoxide dismutase (SOD) activity was significantly increased in the ipsilateral hemisphere of LIF-treated rats compared to rats that received PBS treatment at 72 h after MCAO. Western blot and immunohistochemical analysis revealed that SOD3 was upregulated in brain tissue and induced specifically in cortical neurons tissue at this time point. Neurons that expressed high levels of SOD3 at 72 h after MCAO also showed high levels of phosphorylated Akt (Ser473). LIF (200 ng/ml) reduced necrotic and apoptotic cell death against 24 h of OGD as measured by lactate dehydrogenase (LDH) release and caspase-3 activation. Quantitative real-time PCR analysis showed that LIF treatment upregulated SOD3 gene expression in vitro during OGD. Treatment with 10 µM Akt Inhibitor IV and transfection with SOD3 siRNA counteracted the neuroprotective effects of LIF in vitro, showing that upregulation of SOD3 and activation of Akt signaling are necessary for LIF-mediated neuroprotection. Several transcription factors that regulated Akt-inducible genes were previously identified by this lab, including myeloid zinc finger-1 (MZF-1) and specificity protein-1 (Sp1). The goal of the second set of experiments was to determine whether LIF exerted protective actions through MZF-1 and Sp1. According to analysis with Genomatix, MZF-1 and Sp1 have multiple binding sites in the promoter for the rat SOD3 gene. Western blot analysis showed that there was a trend towards increased MZF-1 protein expression in the brains of LIF-treated rats that approached significance. Immunohistochemical analysis and quantitative real-time PCR showed a significant in vitro upregulation in MZF-1 expression among LIF-treated neurons compared to PBS-treated neurons. Sp1 gene expression was not changed by LIF treatment, but there was a trend towards increased protein expression. In addition, there was a significant correlation between Sp1 and MZF-1 among brain samples from LIF-treated rats but not PBS-treated or sham rats at 72 h after MCAO. Immunohistochemical analysis revealed that Sp1 and MZF-1 co-localized with neuronal nuclei and SOD3 at 72 h after MCAO. Neurons that were transfected with MZF-1 or Sp1 siRNA following isolation did not show a significant decrease in LDH release after 24 h OGD that was observed among neurons transfected with scrambled siRNA. These data demonstrate that Sp1 and MZF-1 are involved with the neuroprotective signaling of LIF under ischemia. This laboratory has demonstrated that LIF activates transcription of protective genes and increases the activity of transcription factors through modulation of intracellular signaling. However, the upstream signaling mechanisms of LIF during ischemia had not previously been investigated. Previous investigators found that the LIF-specific subunit of the heterodimeric LIF receptor (LIFR) is induced by CNS injury. Western blot analysis was used to determine whether LIFR was induced in the brain and the spleen, which plays a role in the peripheral immune response, after MCAO. According to these results, LIF treatment significantly upregulates LIF in the brain compared to PBS treatment or sham injury at 72 h after MCAO. Genomatix analysis of the LIFR promoter region revealed a binding site for Sp1, which is one of the transcription factors responsible for neuroprotection by LIF. At this same time point, splenic LIFR expression is significantly reduced after MCAO compared to sham injury. LIF treatment did not significantly increase LIFR expression, but did significantly increase spleen size compared to PBS treatment at 72 h after MCAO. Although there was a trend towards increased LIFR expression in the spleen from 24 h to 72 h after MCAO, this increase was not statistically significant. However, there was a significant positive correlation between spleen weight and LIFR expression among rats euthanized 24-72 h after MCAO/sham injury. In addition, there was a significant negative correlation between LIFR expression in the brain and the spleen weight, thus showing that LIFR is upregulated following the splenic response. According to findings from other groups, JAK1 has been shown to associate with the heterodimeric LIF receptor (LIFR/gp130) and directly activate PI3K/Akt signaling. To test whether JAK1 contributes neuroprotection during ischemia, cultured neurons were treated with several concentrations (2.5-50 nM) of GLPG0634, a JAK1-specific inhibitor prior to 24 h of OGD. With the exception of the 2.5 nM concentration, all concentrations of GLPG0634 significantly decreased LDH release compared to DMSO treatment, with the 5 nM concentration having the most potent effect on reducing cytotoxicity. However, the 5 nM concentration had no significant did not significantly reduce LDH release compared to DMSO treatment under 24 h of normoxic conditions. These results indicate that JAK1 activity is primarily detrimental to neurons during ischemia. Although it is possible that LIF signaling activates JAK1, it is unlikely that JAK1 is responsible for LIF-mediated neuroprotection during ischemia. The results of these experiments allowed us to determine several molecular mechanisms for LIF-mediated neuroprotection. LIF, which binds to its heterodimeric receptor, activates Akt signaling during ischemia. The transcription factors Sp1 and MZF-1, which are located downstream of Akt, bind to the promoter of the SOD3 gene. In addition, Sp1 also regulates the LIFR gene. SOD3 upregulation increases total SOD activity, which decreases apoptotic and necrotic cell death during apoptosis. Due to its ability to promote antioxidant expression and survival signaling in multiple neural cell types, LIF shows promise as a novel treatment for permanent focal cerebral ischemia.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22073号 / 農博第2365号 / 新制||農||1072(附属図書館) / 学位論文||R1||N5227(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 保川 清, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22510号 / 農博第2414号 / 新制||農||1078(附属図書館) / 学位論文||R2||N5290(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 佐々木 努, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

All cells and organisms consume energy for survival. A robust system has evolved in vertebrates to serve as an energy reservoir. In particular, specialized cells, adipocytes, are responsible for the dynamic storage of energy by accumulating and releasing fatty acids. Fluctuating energy demands require adipose tissue to adjust in size, however complications can arise in both extremes giving rise to systemic diseases, such as obesity and diabetes mellitus (T2D). In mammals, leptin production in adipocytes is up-regulated by feeding and insulin to provide long-term post-prandial satiety. Although this regulatory connection is central to all physiological effects of leptin, the molecular mechanism remains unknown for leptin production. Here, we show that the transcription factor Egr1 is rapidly but transiently induced by insulin in adipose cells both in vitro and in vivo in a mTORC1-dependent fashion. Induction of Egr1 was immediately followed by an increase in leptin transcription. Chromatin immunoprecipitation and luciferase assays demonstrate that Egr1 directly binds to and activates the leptin promoter. Interestingly, the lipid droplet protein Fat specific protein 27 (FSP27) may work as a co-factor for Egr1 in regulating leptin expression. By using siRNA-mediated knock out of Egr1 along with its over-expression in adipocytes, we demonstrate that Egr1 is both necessary and sufficient for the stimulatory effect of insulin on leptin transcription. Knockout of the mTORC1-regulated translation repressor 4EBP1/2 increases leptin transcription both in vitro and in vivo. Adipose specific doxycycline-inducible constitutively active Rheb transgenic mouse lines contained higher circulating leptin and transcription of leptin following doxycycline treatment and were able to maintain elevated leptin levels following a 16 hour fast. Thus, insulin and nutrients, such as amino acids and glucose, activate leptin expression via the mTORC1-Egr1 regulatory axis.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19785号 / 農博第2181号 / 新制||農||1042(附属図書館) / 学位論文||H28||N5001(農学部図書室) / 32821 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 金本 龍平, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

No description available.

G-protein coupled receptor signaling (GPCR) is essential for regulating a large variety of hormonal, sensory and neuronal processes in eukaryotic cells. Because the regulation of these physiological responses is critical, GPCR signaling pathways are carefully controlled at different levels within the cascade. Phosducin-like protein 1 (PhLP1) can bind the G protein βγ dimer and participate in GPCR signaling. Recent evidence has supported the concept that PhLP1 can serve as a co-chaperone of the eukaryotic cytosolic chaperonin complex CCT/TRiC to mediate G βγ assembly. Although a general mechanism of PhLP1-mediated G βγ assembly has been postulated, many of the details about this process are still missing. Structural analysis of key complexes that are important intermediates in the G βγ assembly process can generate snapshots that provide molecular details of the mechanism beyond current understanding. We have isolated two important intermediates in the assembly process, the Gβ1-CCT and PhLP1-Gβ1-CCT complexes assembled in vivo in insect cells, and have determined their structures by cryo-electron microscopy (cryo-EM). Structural analysis reveals that Gβ1, representing the WD40 repeat proteins which are a major class of CCT substrates, interacts specifically with the apical domain of CCTβ. Gβ1 binding experiments with several chimeric CCT subunits confirm a strong interaction of Gβ1 with CCTβ and map Gβ1 binding to α-Helix 9 and the loop between β-strands 6 and 7. These regions are part of a hydrophobic surface of the CCTβ apical domain facing the chaperonin cavity. Docking the Gβ molecule into the two 3D reconstructions (Gβ1-CCT and PhLP1-Gβ1-CCT) reveals that upon PhLP1 binding to Gβ1-CCT, the quasi-folded Gβ molecule is constricted to a more native state and shifted to an angle that can lead to the release of folded Gβ1 from CCT. Moreover, mutagenesis of the CCTβ subunit suggests that PhLP1 can interact with the tip of the apical domain of CCTβ subunit at residue S260, which is a downstream phosphorylation target site of RSK and S6K kinases from the Ras-MAPK and mTOR pathways. These results reveal a novel mechanism of PhLP1-mediated Gβ folding and its release from CCT. The next important step in testing the PhLP1-mediated Gβγ assembly hypothesis is to investigate the function of PhLP1 in vivo. We have prepared a rod-specific PhLP1 conditional knockout mouse in which the physiological consequences of the loss of PhLP1 functions have been characterized. The loss of PhLP1 has led to profound consequences on the ability of these rods to detect light as a result of a significant reduction in the expression of transducin (Gt) subunits. Expression of other G protein subunits as well as Gβ5-RGS9-1 complexes was also greatly decreased, yet all of this occurs without resulting in rapid degeneration of the photoreceptor cells. These results show for the first time the essential nature of PhLP1 for Gβγ and Gβ5-RGS dimer assembly in vivo, confirming results from cell culture and structural studies.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24911号 / 農博第2574号 / 新制||農||1102(附属図書館) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 舟場 正幸, 教授 太田 毅, 教授 横井 伯英 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM