A protocol for rat in vitro fertilization during conventional laboratory working hours

In vitro fertilization (IVF) is a valuable technique for the propagation of experimental animals. IVF has typically been used in mice to rapidly expand breeding colonies and create large numbers of embryos. However, applications of IVF in rat breeding experiments have stalled due to the inconvenient laboratory work schedules imposed by current IVF protocols for this species. Here, we developed a new rat IVF protocol that consists of experimental steps performed during common laboratory working hours. Our protocol can be completed within 12 h by shortening the period of sperm capacitation from 5 to 1 h and the fertilization time from 10 to 8 h in human tubal fluid (HTF) medium. This new protocol generated an excellent birth rate and was applicable not only to closed colony rat strains, such as Wistar, Long-Evans, and Sprague–Dawley (SD), but also to the inbred Lewis strain. Moreover, Wistar and Long-Evans embryos prepared by this protocol were successfully frozen by vitrification and later successfully thawed and resuscitated. This protocol is practical and can be easily adopted by laboratory workers.     

Midday depression of tree root respiration in relation to leaf transpiration

The root respiration rate often shows an exponential or a linear relationship with temperature under laboratory conditions. However, under intact conditions in the field, the root respiration rates of some tree species decreased around midday despite an increment of the root temperature (Bekku et al. 2009). To clarify the cause of midday depression, we examined the relationships between the intact root respiration and parameters of leaf gas exchange through the simultaneous field measurement of the gas exchange in the leaf and root of Quercus crispula and Chamaecyparis obtusa, which are canopy trees. There were no significant relationships between the root respiration rates (R _r) and the parameters of leaf gas exchange in the field. However, in C. obtusa, the relationships between R _r and the transpiration rates (E) at 1 h before the measurement of R _r were fitted by logarithmic function with a determination coefficient of 0.60–0.89. In the light-manipulation experiments using saplings, R _r had significant positive correlations with E at 20 min before the measurement of R _r, root temperature (T _r), and the photosynthesis (P _n) at 20 min before the measurement of R _r. We examined which factor, P _n or E, affects the root respiration rate through a manipulation experiment using a growth chamber regulating the ambient CO₂ concentration and relative humidity independently under constant air temperature and photosynthetic photon flux density. As a result, the root respiration rates changed corresponding to E and not P _n. These results suggest that the root respiration rate of trees changes significantly in the daytime and is affected by the leaf transpiration rate as well as the temperature.     

Photostability of lycopene dispersed in an aqueous solution

Lycopene dispersed in aqueous solutions with different dissolved oxygen contents was photo-irradiated by using a xenon weather meter, and the contents of lycopene and dissolved oxygen were measured. Both the degradation of lycopene and the consumption of dissolved oxygen followed a first-order kinetics model. There was a proportional relationship between the degradation content of lycopene and the consumption of dissolved oxygen. These results indicate that dissolved oxygen would also be involved in the photolysis of lycopene.     

Coordinated regulation of differentiation and proliferation of embryonic cardiomyocytes by a jumonji (Jarid2)-cyclin D1 pathway

In general, cell proliferation and differentiation show an inverse relationship, and are regulated in a coordinated manner during development. Embryonic cardiomyocytes must support embryonic life by functional differentiation such as beating, and proliferate actively to increase the size of the heart. Therefore, progression of both proliferation and differentiation is indispensable. It remains unknown whether proliferation and differentiation are related in these embryonic cardiomyocytes. We focused on abnormal phenotypes, such as hyperproliferation, inhibition of differentiation and enhanced expression of cyclin D1 in cardiomyocytes of mice with mutant jumonji (Jmj, Jarid2), which encodes the repressor of cyclin D1. Analysis of Jmj/cyclin D1 double mutant mice showed that Jmj was required for normal differentiation and normal expression of GATA4 protein through cyclin D1. Analysis of transgenic mice revealed that enhanced expression of cyclin D1 decreased GATA4 protein expression and inhibited the differentiation of cardiomyocytes in a CDK4/6-dependent manner, and that exogenous expression of GATA4 rescued the abnormal differentiation. Finally, CDK4 phosphorylated GATA4 directly, which promoted the degradation of GATA4 in cultured cells. These results suggest that CDK4 activated by cyclin D1 inhibits differentiation of cardiomyocytes by degradation of GATA4, and that initiation of Jmj expression unleashes the inhibition by repression of cyclin D1 expression and allows progression of differentiation, as well as repression of proliferation. Thus, a Jmj-cyclin D1 pathway coordinately regulates proliferation and differentiation of cardiomyocytes.     

Xanthoangelols isolated from Angelica keiskei inhibit inflammatory-induced plasminogen activator inhibitor 1 (PAI-1) production

The folk medicine Angelica keiskei (Ashitaba) exhibits antitumor, antioxidant and antidiabetic activities and it has recently attracted attention as a health food. Ashitaba is thought to have antithrombotic properties, but this has not yet been scientifically proven. The elevation of plasma plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis results in a predisposition to the risk of thrombosis. The present study showed that Ashitaba exudates injected intraperitoneally and orally administered over long-term suppressed the lipopolysaccharide (LPS) induced PAI-1 increase in mouse plasma. We also found that xanthoangelol, xanthoangelols B and D, the components of Ashitaba exudates, significantly inhibited TNFα-induced PAI-1 production from human umbilical vein endothelial cells (HUVECs). These findings suggest that Ashitaba can decrease elevated PAI-1 production, and that daily consumption of Ashitaba product might maintain anticoagulant status by inhibiting elevations in PAI-1 under inflammatory conditions.     

Characterization of photosystem II assembly complexes containing ONE-HELIX PROTEIN1 in Arabidopsis thaliana

Journal of Plant Research - The assembly process of photosystem II (PSII) requires several auxiliary proteins to form assembly intermediates. In plants, early assembly intermediates comprise D1 and...     

Immunosuppressive effect of a non-proteinogenic amino acid from Streptomyces through inhibiting allogeneic T cell proliferation

The immunosuppressive activity of myriocin (ISP-1), a lead compound of fingolimod (FTY720), is derived from its 2-amino-1,3-propandiol structure. A non-proteinogenic amino acid, (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid (DADH), that contains this structure, was recently identified as a biosynthetic intermediate of a dipeptide secondary metabolite, vazabitide A, in Streptmyces sp. SANK 60404; however its effect on adaptive immunity has not yet been examined. In this study, we examined whether DADH suppresses mixed lymphocyte reaction using mouse bone marrow-derived dendritic cells (BMDCs) and allogeneic splenic T cells. Although T cell proliferation induced by cross-linking CD3 and CD28 were not suppressed by DADH unlike ISP-1, the pre-incubation of BMDCs with DADH but not ISP-1 significantly decreased allogeneic CD8⁺ T cell expansion. Based on these results, we concluded that DADH suppresses DC-mediated T cell activation by targeting DCs.     

Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.