Gel to Liquid-Crystalline Phase Transitions of Lipids and Membranes Isolated from Escherichia coli Cells

Differential scanning calorimetry (DSC) was used to examine the relationship of the gel to liquid-crystalline phase transition of lipids to fatty acid composition with membrane lipids and spheroplast membranes isolated from cells of a wild strain and an unsaturated fatty acid auxotroph of Escherichia coli grown under various conditions. These lipids and membranes underwent thermotropic phase transitions at different temperatures depending on the thermal properties of their constituent fatty acids. The lipid phase transition occurred at higher temperatures in biomembranes than in extracted lipids. DSC thermograms of lipids synthesized by bacterial cells which were observed at a temperature scanning rate as slow as 0.3 K min^-1 were characterized by a distinctly plain peak summit. Endothermic peaks given by samples derived from elaidic acid-enriched cells were relatively narrow and asymmetric. The discrepancy between the transition temperatures measured with extracted lipids and with membraneous fractions, and the shape of the endothermic peaks, are discussed.     

Novel post-column derivatization method for the fluorimetric determination of norepinephrine and epinephrine

A novel method is described in which catecholamines are converted into fluorescent products by heating in alkaline borate buffer. The method was applied to the determination of norepinephrine and epinephrine after separation by high-performance liquid chromatography using a pellicular, strong cation exchanger. The new system is simpler than the system based on the trihydroxyindole reaction. It is suitable for the measurement of catecholamines in the range 0.25–20 ng. The assay of catecholamines in human urine is also described.     

Rapid degeneration of the protoplasm in artificially induced small cells ofMicrasterias crux melitensis

Summary Growing cells ofMicrasterias crux melitensis grew into asymmetric cells with small half cells in 0.2 M mannitol. Cells with small half cells could multiply in the normal culture medium. When mother half cells were less than half the size of the normal mother half cells small daughter half cells were formed. When the mother half cells were very small and had no chloroplasts, the protoplasts rapidly degenerated. Many autophagic vacuoles and lysosomes were found in the protoplasm of the degenerating cells. The rapid degeneration was assumed to be due to the activity of these autophagic lysosomes, whose induction is probably triggered by some factor produced by the shortage of protein precursors and energy sources.     

Automated Software Analysis of Fetal Movement Recorded during a Pregnant Woman’s Sleep at Home

Fetal movement is an important biological index of fetal well-being. Since 2008, we have been developing an original capacitive acceleration sensor and device that a pregnant woman can easily use to record fetal movement by herself at home during sleep. In this study, we report a newly developed automated software system for analyzing recorded fetal movement. This study will introduce the system and compare its results to those of a manual analysis of the same fetal movement signals (Experiment I). We will also demonstrate an appropriate way to use the system (Experiment II). In Experiment I, fetal movement data reported previously for six pregnant women at 28-38 gestational weeks were used. We evaluated the agreement of the manual and automated analyses for the same 10-sec epochs using prevalence-adjusted bias-adjusted kappa (PABAK) including quantitative indicators for prevalence and bias. The mean PABAK value was 0.83, which can be considered almost perfect. In Experiment II, twelve pregnant women at 24-36 gestational weeks recorded fetal movement at night once every four weeks. Overall, mean fetal movement counts per hour during maternal sleep significantly decreased along with gestational weeks, though individual differences in fetal development were noted. This newly developed automated analysis system can provide important data throughout late pregnancy.     

Genome-Wide Screening of Salt Tolerant Genes by Activation-Tagging Using Dedifferentiated Calli of Arabidopsis and Its Application to Finding Gene for Myo-Inositol-1-P-Synthase

Salinity represents a major abiotic stress factor that can adversely limit the production, quality and geographical distribution of crops. In this study we focused on dedifferentiated calli with fundamental cell functions, the salt tolerance of which had not been previously examined. The experimental approach was based on activation tagging without regeneration of plants for the identification of salt-tolerant mutants of Arabidopsis. Among 62,000 transformed calli that were screened, 18 potential mutants resistant to 150 mM NaCl were obtained. Thermal asymmetric interlaced (TAIL)-PCR was performed to determine the location of T-DNA integration in the genome. In one line, referred to as salt tolerant callus 1 (stc1), expression of a gene [At4g39800: myo-inositol-1-P-synthase 1 (MIPS1)] was considerably enhanced in calli. Plants regenerated from calli showed tolerance to salt in germination and subsequent growth. Retransformation of wild-type Arabidopsis with MIPS1 conferred salt tolerance, indicating that MIPS1 is the causal gene. The over-expression of MIPS1 increased the content of total inositol. The involvement of MIPS1 in salt tolerance through the fundamental cell growth has been proved in Arabidopsis.     

Visual Cycle Modulation as an Approach toward Preservation of Retinal Integrity

Increased exposure to blue or visible light, fluctuations in oxygen tension, and the excessive accumulation of toxic retinoid byproducts places a tremendous amount of stress on the retina. Reduction of visual chromophore biosynthesis may be an effective method to reduce the impact of these stressors and preserve retinal integrity. A class of non-retinoid, small molecule compounds that target key proteins of the visual cycle have been developed. The first candidate in this class of compounds, referred to as visual cycle modulators, is emixustat hydrochloride (emixustat). Here, we describe the effects of emixustat, an inhibitor of the visual cycle isomerase (RPE65), on visual cycle function and preservation of retinal integrity in animal models. Emixustat potently inhibited isomerase activity in vitro (IC₅₀ = 4.4 nM) and was found to reduce the production of visual chromophore (11-cis retinal) in wild-type mice following a single oral dose (ED₅₀ = 0.18 mg/kg). Measure of drug effect on the retina by electroretinography revealed a dose-dependent slowing of rod photoreceptor recovery (ED₅₀ = 0.21 mg/kg) that was consistent with the pattern of visual chromophore reduction. In albino mice, emixustat was shown to be effective in preventing photoreceptor cell death caused by intense light exposure. Pre-treatment with a single dose of emixustat (0.3 mg/kg) provided a ~50% protective effect against light-induced photoreceptor cell loss, while higher doses (1–3 mg/kg) were nearly 100% effective. In Abca4-/- mice, an animal model of excessive lipofuscin and retinoid toxin (A2E) accumulation, chronic (3 month) emixustat treatment markedly reduced lipofuscin autofluorescence and reduced A2E levels by ~60% (ED₅₀ = 0.47 mg/kg). Finally, in the retinopathy of prematurity rodent model, treatment with emixustat during the period of ischemia and reperfusion injury produced a ~30% reduction in retinal neovascularization (ED₅₀ = 0.46mg/kg). These data demonstrate the ability of emixustat to modulate visual cycle activity and reduce pathology associated with various biochemical and environmental stressors in animal models. Other attributes of emixustat, such as oral bioavailability and target specificity make it an attractive candidate for clinical development in the treatment of retinal disease.     

Cryptococcus neoformans Infection in Mice Lacking Type I Interferon Signaling Leads to Increased Fungal Clearance and IL-4-Dependent Mucin Production in the Lungs

Type I interferons (IFNs) are secreted by many cell types upon stimulation via pattern recognition receptors and bind to IFN-α/β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2. Although type I IFNs are well known as anti-viral cytokines, limited information is available on their role during fungal infection. In the present study, we addressed this issue by examining the effect of IFNAR1 defects on the host defense response to Cryptococcus neoformans. In IFNAR1KO mice, the number of live colonies was lower and the host immune response mediated not only by Th1 but also by Th2 and Th17-related cytokines was more accelerated in the infected lungs than in WT mice. In addition, mucin production by bronchoepithelial cells and expression of MUC5AC, a major core protein of mucin in the lungs, were significantly higher in IFNAR1KO mice than in WT mice. This increase in mucin and MUC5AC production was significantly inhibited by treatment with neutralizing anti-IL-4 mAb. In contrast, administration of recombinant IFN-αA/D significantly suppressed the production of IL-4, but not of IFN-γ and IL-17A, in the lungs of WT mice after cryptococcal infection. These results indicate that defects of IFNAR1 led to improved clearance of infection with C. neoformans and enhanced synthesis of IFN-γ and the IL-4-dependent production of mucin. They also suggest that type I IFNs may be involved in the negative regulation of early host defense to this infection.