Synthesis and evaluation of a selective molecularly imprinted polymer for the contraceptive drug levonorgestrel

A molecularly imprinted polymer (MIP) has been prepared using levonorgestrel (LEV) as template. The polymer was synthesised in a non-covalent approach using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linking monomer via a free radical polymerization. An equivalent blank polymer was also synthesised in the absence of the template compound. Batch adsorption experiments were used to evaluate the binding affinity of the imprinted polymer. After packing MIP into a stainless steel column (150 mm x 4.6 mm i.d.), retention and elution of the template and related compounds were evaluated by high-performance liquid chromatography (HPLC). This LEV imprinted polymer was further applied for selective solid phase extraction (SPE) of LEV from human serum. It was confirmed that the binding ability of the prepared MIP for LEV was essentially sufficient in the presence of other compounds coexisting in serum sample. Therefore, as a selective and efficient solid phase material, LEV imprinted polymer has a high potential application in analysis of this steroidal hormone in clinical purposes.     

Microbial community analysis of field-grown soybeans with different nodulation phenotypes

Microorganisms associated with the stems and roots of nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod(+) soybean roots. Fusarium solani was stably associated with nodulated (Nod(+) and Nod(++)) roots and less abundant in Nod(-) soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod(-), Nod(+), or Nod(++)). The analysis of root samples indicated that the microbial community in Nod(-) soybeans was more similar to that in Nod(++) soybeans than to that in Nod(+) soybeans.     

Characterization of glutamate decarboxylase from a high gamma-aminobutyric acid (GABA)-producer, Lactobacillus paracasei

Gamma-aminobutyric acid (GABA) has several physiological functions in humans. We have reported that Lactobacillus paracasei NFRI 7415 produces high levels of GABA. To gain insight into the higher GABA-producing ability of this strain, we analyzed glutamate decarboxylase (GAD), which catalyzes the decarboxylation of L-glutamate to GABA. The molecular weight of the purified GAD was estimated to be 57 kDa by SDS-PAGE and 110 kDa by gel filtration, suggesting that GAD forms the dimer under native conditions. GAD activity was optimal at pH 5.0 at 50 degrees C. The Km value for the catalysis of glutamate was 5.0 mM, and the maximum rate of catalysis was 7.5 micromol min(-1) mg(-1). The N-terminal amino acid sequence of GAD was determined, and the gene encoding GAD from genomic DNA was cloned. The findings suggest that the ability of Lb. paracasei to produce high levels of GABA results from two characteristics of GAD, viz., a low Km value and activity at low pH.     

Identification of a plasmin-interactive site within the A2 domain of the factor VIII heavy chain

Factor VIII is activated and inactivated by plasmin by limited proteolysis. In our one-stage clotting assay, these plasmin-catalyzed reactions were inhibited by the addition of isolated factor VIII A2 subunits and by Glu-Gly-Arg-active-site modified factor IXa. SDS-PAGE analysis showed that an anti-A2 monoclonal antibody, recognizing the factor IXa-interactive site (residues 484-509), blocked the plasmin-catalyzed cleavage at Arg(336) and Arg(372) but not at Arg(740). Surface plasmon resonance-based assays and ELISA demonstrated that the A2 subunit bound to active-site modified anhydro-plasmin with high affinity (K(d): 21 nM). Both an anti-A2 monoclonal antibody and a peptide comprising of A2 residues 479-504 blocked A2 binding by approximately 80% and approximately 55%, respectively. Mutant A2 molecules where the basic residues in A2 were converted to alanine were evaluated for binding of anhydro-plasmin. Among the tested mutants, the R484A A2 mutant possessed approximately 250-fold lower affinity than the wild-type A2. The affinities of K377A, K466A, and R471A mutants were decreased by 10-20-fold. The inhibitory effect of R484A mutant on plasmin-catalyzed inactivation of factor VIIIa was approximately 20% of that of wild-type A2. In addition, the inactivation rate by plasmin of factor VIIIa reconstituted with R484A mutant was approximately 3-fold lower than that with wild-type A2. These findings demonstrate that Arg(484) plays a key role within the A2 plasmin-binding site, responsible for plasmin-catalyzed factor VIII(a) inactivation.     

Effect of cold stimulation during slow-wave sleep on sleep cycle

To clarify the effect of cold stimulation during slow-wave sleep (SWS) on the sleep cycle, we conducted a sleep experiment. Five healthy males slept on a bedding system we developed to make the inside of bedding cooler. When the subject was sleeping deeply in the second and fourth SWS, the system cooled their bedding. When the subject's sleep condition shifted toward arousal, the cold air was stopped. As a result, all subjects’ sleep stage shifted to light sleep and reached arousal. After stopping stimulation, they immediately returned to the SWS at the first stimulation. But at the second stimulation, the sleep state did not return to the SWS.     

Self-Cloning Baker's Yeasts That Accumulate Proline Enhance Freeze Tolerance in Doughs

We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding γ-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking.     

Intermittent hypoxia increases ventilation and Sa(O2) during hypoxic exercise and hypoxic chemosensitivity.

The purpose of this study was 1) to test the hypothesis that ventilation and arterial oxygen saturation (Sa(O2)) during acute hypoxia may increase during intermittent hypoxia and remain elevated for a week without hypoxic exposure and 2) to clarify whether the changes in ventilation and Sa(O2) during hypoxic exercise are correlated with the change in hypoxic chemosensitivity. Six subjects were exposed to a simulated altitude of 4,500 m altitude for 7 days (1 h/day). Oxygen uptake (VO2), expired minute ventilation (VE), and Sa(O2) were measured during maximal and submaximal exercise at 432 Torr before (Pre), after intermittent hypoxia (Post), and again after a week at sea level (De). Hypoxic ventilatory response (HVR) was also determined. At both Post and De, significant increases from Pre were found in HVR at rest and in ventilatory equivalent for O2 (VE/VO2) and Sa(O2) during submaximal exercise. There were significant correlations among the changes in HVR at rest and in VE/VO2 and Sa(O2) during hypoxic exercise during intermittent hypoxia. We conclude that 1 wk of daily exposure to 1 h of hypoxia significantly improved oxygenation in exercise during subsequent acute hypoxic exposures up to 1 wk after the conditioning, presumably caused by the enhanced hypoxic ventilatory chemosensitivity.     

Genomic organization and expression of the doublesex-related gene cluster in vertebrates and detection of putative regulatory regions for DMRT1.

Genes related to the Drosophila melanogaster doublesex and Caenorhabditis elegans mab-3 genes are conserved in human. They are identified by a DNA-binding homology motif, the DM domain, and constitute a gene family (DMRTs). Unlike the invertebrate genes, whose role in the sex-determination process is essentially understood, the function of the different vertebrate DMRT genes is not as clear. Evidence has accumulated for the involvement of DMRT1 in male sex determination and differentiation. DMRT2 (known as terra in zebrafish) seems to be a critical factor for somitogenesis. To contribute to a better understanding of the function of this important gene family, we have analyzed DMRT1, DMRT2, and DMRT3 from the genome model organism Fugu rubripes and the medakafish, a complementary model organism for genetics and functional studies. We found conservation of synteny of human chromosome 9 in F. rubripes and an identical gene cluster organization of the DMRTs in both fish. Although expression analysis and gene linkage mapping in medaka exclude a function for any of the three genes in the primary step of male sex determination, comparison of F. rubripes and human sequences uncovered three putative regulatory regions that might have a role in more downstream events of sex determination and human XY sex reversal.     

Impaired autophagy by soluble endoglin,under physiological hypoxia in early pregnant period,is involved in poor placentation in preeclampsia

In early pregnancy, trophoblasts and the fetus experience hypoxic and low-nutrient conditions; nevertheless, trophoblasts invade the uterine myometrium up to one third of its depth and migrate along the lumina of spiral arterioles, replacing the maternal endothelial lining. Here, we showed that autophagy, an intracellular bulk degradation system, occurred in extravillous trophoblast (EVT) cells under hypoxia in vitro and in vivo. An enhancement of autophagy was observed in EVTs in early placental tissues, which suffer from physiological hypoxia. The invasion and vascular remodeling under hypoxia were significantly reduced in autophagy-deficient EVT cells compared with wild-type EVT cells. Interestingly, soluble endoglin (sENG), which increased in sera in preeclamptic cases, suppressed EVT invasion by inhibiting autophagy. The sENG-inhibited EVT invasion was recovered by TGFB1 treatment in a dose-dependent manner. A high dose of sENG inhibited the vascular construction by EVT cells and human umbilical vein endothelial cells (HUVECs), meanwhile a low dose of sENG inhibited the replacement of HUVECs by EVT cells. A protein selectively degraded by autophagy, SQSTM1, accumulated in EVT cells in preeclamptic placental biopsy samples showing impaired autophagy. This is the first report showing that impaired autophagy in EVT contributes to the pathophysiology of preeclampsia.     

Accuracy evaluation of sleep-wake stage analysis with SleepSign Ver2.0

Sleep and Biological Rhythms -