High-performance liquid chromatographic method for measurement of cytochrome P450-mediated metabolism of 7-ethoxy-4-trifluoromethylcoumarin

An HPLC method for analysis of deethylation of 7-ethoxy-4-trifluoromethylcoumarin (ETFMC), a substrate of various enzymes of the cytochrome P450 superfamily, was developed. ETFMC was incubated at 37°C with human hepatic microsomes or microsomes prepared from a lymphoblastoid cell line that expresses human CYP2B6. Under these conditions, the highly fluorescent metabolite 7-hydroxy-4-trifluoromethylcoumarin (HTFMC) is formed. The metabolite was analyzed by reversed-phase HPLC with fluorescence detection. The limits of detection of the metabolite were 5.0 fmol per injection, a sensitivity at least one order of magnitude greater than the standard method, which does not involve HPLC. This method will be of great utility when quantities of microsomal protein from cell lines expressing human CYP enzymes are limited.     

Expression and regulation of hFIX minigene and cDNA driven by β-casein gene in mouse mammary gland

Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β-casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and Lac2 gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ-carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β-casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β-casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector. Project supported by the State High Technology Development Program and Shanghai Science and Technology Commission.     

GERMINATION ECOPHYSIOLOGY OF HYDROPHYLLUM APPENDICULATUM,A MESIC FOREST BIENNIAL

In north central Kentucky, seeds of the mesic forest biennial Hydrophyllum appendiculatum Michx., are innately dormant at maturity in June. Under natural and simulated seasonal temperature changes, dormancy break occurred in two stages. Root dormancy was broken by high summer temperatures, and shoot dormancy was broken by low winter temperatures. Consequently, roots emerged from seeds during autumn, and cotyledons emerged the following spring. A 90-day warm (30/15 C) stratification treatment broke root dormancy, but the roots emerged only after transfer to lower temperatures. After the warm stratification treatment, roots emerged from 93, 73, 6 and 9% of the seeds incubated at 5, 15/6, 20/10 and 30/15 C (12/12 hr), respectively. Zero, 28, 56 and 84 days of cold (5 C) stratification of seeds with emerged roots resulted in 9, 21, 49 and 82% cotyledon emergence, respectively, at 20/10 C. Thus, H. appendiculatum exhibits a type of morpho-physiological dormancy known as epicotyl dormancy. Although many seeds germinate the first year, others remain dormant and germinate in successive years until the fourth season after ripening.     

Identification of differentially methylated markers among cytogenetic risk groups of acute myeloid leukemia

Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as MN1, UHRF1, HOXB3, and HOXB4, as well as TRIM71, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at HOXB3 and HOXB4 was associated with increased gene expression of both HOXB genes specific to the mid-risk AML, while increased DNA methylation at DCC distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.     

Interactions with successional stage and nutrient status determines the life‐form‐specific effects of increased soil temperature on boreal forest floor vegetation

The boreal forest is one of the largest terrestrial biomes and plays a key role for the global carbon balance and climate. The forest floor vegetation has a strong influence on the carbon and nitrogen cycles of the forests and is sensitive to changes in temperature conditions and nutrient availability. Additionally, the effects of climate warming on forest floor vegetation have been suggested to be moderated by the tree layer. Data on the effects of soil warming on forest floor vegetation from the boreal forest are, however, very scarce. We studied the effects on the forest floor vegetation in a long‐term (18 years) soil warming and fertilization experiment in a Norway spruce stand in northern Sweden. During the first 9 years, warming favored early successional species such as grasses and forbs at the expense of dwarf shrubs and bryophytes in unfertilized stands, while the effects were smaller after fertilization. Hence, warming led to significant changes in species composition and an increase in species richness in the open canopy nutrient limited forest. After another 9 years of warming and increasing tree canopy closure, most of the initial effects had ceased, indicating an interaction between forest succession and warming. The only remaining effect of warming was on the abundance of bryophytes, which contrary to the initial phase was strongly favored by warming. We propose that the suggested moderating effects of the tree layer are specific to plant life‐form and conclude that the successional phase of the forest may have a considerable impact on the effects of climate change on forest floor vegetation and its feedback effects on the carbon and nitrogen cycles, and thus on the climate.     

Macular Microcysts in Mitochondrial Optic Neuropathies: Prevalence and Retinal Layer Thickness Measurements

Purpose

To investigate the thickness of the retinal layers and to assess the prevalence of macular microcysts (MM) in the inner nuclear layer (INL) of patients with mitochondrial optic neuropathies (MON).

Methods

All patients with molecularly confirmed MON, i.e. Leber’s Hereditary Optic Neuropathy (LHON) and Dominant Optic Atrophy (DOA), referred between 2010 and 2012 were enrolled. Eight patients with MM were compared with two control groups: MON patients without MM matched by age, peripapillary retinal nerve fiber layer (RNFL) thickness, and visual acuity, as well as age-matched controls. Retinal segmentation was performed using specific Optical coherence tomography (OCT) software (Carl Zeiss Meditec). Macular segmentation thickness values of the three groups were compared by one-way analysis of variance with Bonferroni post hoc corrections.

Results

MM were identified in 5/90 (5.6%) patients with LHON and 3/58 (5.2%) with DOA. The INL was thicker in patients with MON compared to controls regardless of the presence of MM [133.1±7μm vs 122.3±9μm in MM patients (p<0.01) and 128.5±8μm vs. 122.3±9μm in no-MM patients (p<0.05)], however the outer nuclear layer (ONL) was thicker in patients with MM (101.4±1mμ) compared to patients without MM [77.5±8mμ (p<0.001)] and controls [78.4±7mμ (p<0.001)]. ONL thickness did not significantly differ between patients without MM and controls.

Conclusion

The prevalence of MM in MON is low (5-6%), but associated with ONL thickening. We speculate that in MON patients with MM, vitreo-retinal traction contributes to the thickening of ONL as well as to the production of cystic spaces.