Germline recombination in a novel Cre transgenic line,Prl3b1‐Cre mouse

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1‐cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL‐2) protein in placenta along with increased expression toward the end of pregnancy. PL‐2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1‐cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1‐cre;R26GRR mice revealed that tdsRed‐positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1‐cre;R26GRR testes suggested that Cre‐mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1‐cre mice line provides a unique resource to understand testicular germ‐cell development. genesis 54:389–397, 2016. © 2016 Wiley Periodicals, Inc.     

Establishment and characterization of a novel ovarian clear cell carcinoma cell line,TU-OC-2, with loss of ARID1A expression

A new cell line of human ovarian clear cell carcinoma (CCC), TU-OC-2, was established and characterized. The cells were polygonal in shape, grew in monolayers without contact inhibition and were arranged in islands like pieces of a jigsaw puzzle. The chromosome numbers ranged from 41 to 96. A low rate of proliferation was observed and the doubling time was 37.5 h. The IC₅₀ values of cisplatin, 7-ethyl-10-hydroxycamptothecin (SN38), which is an active metabolite of camptothecin, and paclitaxel were 7.7 μM, 17.7 nM and 301 nM, respectively. The drug sensitivity assay indicated that TU-OC-2 was sensitive to SN38, but resistant to cisplatin and paclitaxel. Mutational analysis revealed that TU-OC-2 cells have no mutations of PIK3CA in exons 9 and 20 and of TP53 in exons 4–9. We observed the loss of ARID1A protein expression in TU-OC-2 cells by western blot analysis and in the original tumor tissue by immunohistochemistry. This cell line may be useful for studying the chemoresistant mechanisms of CCC and exploring novel therapeutic targets such as the ARID1A-related signaling pathway.     

The influence of microclimate on bryophyte diversity in an urban Japanese garden landscape

Landscape and Ecological Engineering - Japanese gardens play an important role in the conservation of bryophyte diversity. Previous studies have indicated that diverse garden landscapes and their...     

Fmoc-based solid-phase synthesis of GPR54-agonistic pentapeptide derivatives containing alkene- and fluoroalkene-dipeptide isosteres

Fmoc-protected Phe-Gly-type (Z)-alkene dipeptide isostere (ADI) and (E)-fluoroalkene dipeptide isostere (FADI) were synthesized and applied to Fmoc-based solid-phase peptide synthesis (SPPS). These cis-peptide bond mimetics were introduced into a bioactive pentapeptide [H-Amb-Phe-Gly-Leu-Arg-Trp-NH(2); Amb = 4-(aminomethyl) benzoic acid], which has potent GPR54 agonistic activity. The resulting pentapeptide derivatives showed low GPR54 agonistic activity, as compared with the parent peptide and (E)-ADI-containing derivative. This suggests that the trans-amide conformer of Phe-Gly peptide bond of the parent peptide would be significantly important for bioactivity. Contrary to our expectations, a (Z)-FADI-containing derivative exhibited essentially no activity, revealing the necessity of critical validation of FADI-bioisosterism.     

Thrombomodulin is a clock-controlled gene in vascular endothelial cells

Cardiovascular diseases are closely related to circadian rhythm, which is under the control of an internal biological clock mechanism. Although a biological clock exists not only in the hypothalamus but also in each peripheral tissue, the biological relevance of the peripheral clock remains to be elucidated. In this study we searched for clock-controlled genes in vascular endothelial cells using microarray technology. The expression of a total of 229 genes was up-regulated by CLOCK/BMAL2. Among the genes that we identified, we examined the thrombomodulin (TM) gene further, because TM is an integral membrane glycoprotein that is expressed primarily in vascular endothelial cells and plays a major role in the regulation of intravascular coagulation. TM mRNA and protein expression showed a clear circadian oscillation in the mouse lung and heart. Reporter analyses, gel shift assays, and chromatin immunoprecipitation analyses using the TM promoter revealed that a heterodimer of CLOCK and BMAL2 binds directly to the E-box of the TM promoter, resulting in TM promoter transactivation. Indeed, the oscillation of TM gene expression was abolished in clock mutant mice, suggesting that TM expression is regulated by the clock gene in vivo. Finally, the phase of circadian oscillation of TM mRNA expression was altered by temporal feeding restriction, suggesting TM gene expression is regulated by the peripheral clock system. In conclusion, these data suggest that the peripheral clock in vascular endothelial cells regulates TM gene expression and that the oscillation of TM expression may contribute to the circadian variation of cardiovascular events.     

A Novel Oxazolidine Linker for the Synthesis of Peptide Aldehydes

A reliable method for solid-phase synthesis of peptide aldehydes by using a new oxazolidine linker is described. Based on a comparative study using the usual cleavage protocol as is used for the Fmoc-based peptide synthesis, we found that this new linker is more appropriate for the synthesis of peptide aldehydes compared with the precedent acetal, semicarbazone or threonine linker. Whereas N-Acylated oxazolidines might be partially deprotected to non-N-acylated intermediates in the TFA cocktail containing several soft nucleophiles which cause significant side reactions, the new oxazolidine linker could produce the desired peptide aldehydes by simple Et₂O washing and subsequent aqueous workup in high chemical yields and purity. We demonstrate the new method is useful especially for the preparation of highly functionalized long-chain peptide aldehydes which require several scavenger chemicals in the final deprotection step. This paper is dedicated to the memory of the late Prof. R. Bruce Merrifield, who passed away May 14, 2006.     

Effect of circadian rhythm type on serum lipid levels in shift workers: A 5-year cohort study

We investigated how differences in circadian rhythm type affect the health of workers engaged in shift work. Employees, who were newly hired in a steel company between 2007 and 2011, received the Morningness–Eveningness Questionnaire (MEQ) survey. The target participants were 153 male shift workers who were not being treated with any antihyperlipidemic drugs and underwent periodic physical examinations including blood tests at least twice. According to the score of the MEQ at the time of joining the company, we classified the subjects into five types. Longitudinal changes in serum lipid level were estimated among the circadian rhythm types adjusted for age, BMI, and other covariates using a linear mixed model. The regression coefficient of total cholesterol level in the “definitely and moderately morning” group was ?17.83 (95% confidence interval (CI): ?33.42 to ?2.23), and in the “intermediate ‘group’ was ?16.84 [95% CI: ?30.40 to ?3.28], compared to the moderate evening type.” The total cholesterol level was higher in the moderately evening type than in any of the other groups. Between the Morningness–Eveningness (ME) type and Low-density lipoprotein (LDL) cholesterol levels, compared with the “moderately evening type” group, the regression coefficient in the “intermediate type” group was ?16.08 (95% CI: ?28.79 to ?3.37), and in the “definitely and moderately morning type” group was ?17.50 [95% CI: ?32.11 to ?2.88]. The “moderately evening type” group had a higher LDL cholesterol level than any of the other groups. Evening-type circadian rhythm type shift workers are more prone to elevated serum lipid levels.     

Effects of Rhododendron removal and prescribed fire on bees and plants in the southern Appalachians

Rhododendron maximum is an evergreen shrub native to the Appalachian Mountains of North America that has expanded in recent decades due to past disturbances and land management. The purpose of this study was to explore how bees and plants were affected by the experimental removal of R. maximum followed by a prescribed fire in one watershed compared to a neighboring reference watershed. Bees and plants were sampled for three years in both watersheds. Comparisons were based on the rarefaction and extrapolation sampling curves of Hill numbers as well as multivariate methods to assess effects on community composition. Bee richness, Shannon''s diversity, and Simpson''s diversity did not differ between watersheds in the year after removal but were all significantly higher in the removal watershed in year two, following the prescribed fire. Bee Shannon''s diversity and Simpson''s diversity, but not richness, remained significantly higher in the removal watershed in the third year. Similar but weaker patterns were observed for plants. Comparisons of community composition found significant differences for bees in the second and third year and significant differences for plants in all three years. For both groups, significant indicator taxa were mostly associated with the removal watershed. Because bees appeared to respond more strongly to the prescribed fire than to the removal of R. maximum and these benefits weakened considerably one year after the fire, clearing R. maximum does not appear to dramatically improve pollinator habitat in the southern Appalachians. This conclusion is underscored by the fact that about one quarter of the bee species in our study area were observed visiting R. maximum flowers. The creation of open areas with wildflowers may be a better way to benefit bees in this region judging from the high diversity of bees captured in the small roadside clearings in this study.     

Disruption of phospholipase B gene, PLB1, increases the survival of baker's yeast Torulaspora delbrueckii

Abstract An uracil auxotrophic mutant of baker's yeast Torulaspora delbrueckii , which is resitant to 5-fluoro-orotic acid, was complemented by transformation with YEp24 which harbors 2 μm origin and URA3 derived from Saccharomyces cerevisiae . The phospholipase B in T. delbrueckii cells is active in both acidic and alkaline conditions. However, activity of phospholipase B gene ( PLB1 ) in cells of disruption mutant ( plbI : : URA3 ) was lost in both conditions, which indicates that all phospholipase B activity is encoded by a single gene (or a single polypeptide) in these yeast cells. Over-expression of PLB1 with YEp plasmid vector in T. delbrueckii cells showed ∼ 2.5-fold increase in phospholipase B activity, comparing with that in wild-type cells. Cells of plb1 Δ mutant showed increased survival when cells of plb1 Δ mutant and wild-type strain were incubated in water at 30 °C. Cells of PLB1 -over-expressed strain died rapidly even during the cultivation period, indicating that phospholipase B activity may be a determinant for the survival of this yeast.