Effect of 5-Hydroxytryptamine on Steroidogenesis and Oocyte Maturation in Pre-ovulatory Follicles of the Medaka Oryzias latipes

The effect of 5-hydroxytryptamine (5-HT) on steroidogenesis and oocyte maturation in pre-ovulatory follicles of the medaka Oryzias lalipes was examined using in vitro culture system. The earliest breakdown of the germinal vesicle of intrafollicular oocytes occurred about 17 hr after the beginning of incubation in the presence of 5-HT at concentration of 10 ng/ml or more. 5-HT induced oocyte maturation in a dose-dependent manner. Cyanoketone inhibited this stimulation. The concentration of 5-HT required to induce oocyte maturation corresponded to that required to enhance the production (secretion) of estradiol-17β and 17α,20β-dihydroxy-4-pregnen-3-one by pre-ovulatory follicle cells. At a concentration of 1 μg/ml, the follicle had to be exposed to 5-HT for at least 4 hr for oocyte maturation accompanied by ovulation to occur. These results indicate that 5-HT induces in vitro maturation of medaka oocytes by stimulating 17α,20β-dihydroxy-4-pregnen-3-one production by pre-ovulatory follicular cells.     

Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

We recently reported a novel heme sensor using fluorescently labeled heme oxygenase-1; however, its inherent enzyme activity would be a potential obstacle in quantifying heme in biological samples. Here, we found that mutation of the catalytically important residue, Asp140, with histidine in the sensor not only diminished the heme degradation activity but also increased heme binding affinity. The sensor with a visible fluorophore was also found to be beneficial to avoid background emission from endogenous substance in biological samples. By using the improved heme sensor, we succeeded in quantifying free heme in rat hepatic samples for the first time.     

A homogeneous DNA hybridization system by using a new luminescence terbium chelate

Homogeneous DNA hybridization assay based on the luminescence resonance energy transfer (LRET) from a new luminescence terbium chelate, N,N,N(1),N(1)-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA)-Tb(3+) (lambda(ex) = 325 nm and lambda(em) = 545 nm) to an organic dye, Cy3 (lambda(ex) = 548 nm and lambda(em) = 565 nm), has been developed. In the system, two DNA probes whose sequences are complementary to the two different consecutive sequences of a target DNA are used; one of the probes is labeled with the Tb(3+) chelate at the 3'-end, and the other is with Cy3 at the 5'-end. Labeling of the Tb(3+) chelate is accomplished via the linkage of a biotin-labeled DNA probe with the Tb(3+) chelate-labeled streptavidin. Strong sensitized emission of Cy3 was observed upon excitation of the Tb(3+) chelate at 325 nm, when the two probe DNAs were hybridized with the target DNA. The sensitivity of the assay was very high compared with those of the previous homogeneous-format assays using the conventional organic dyes; the detection limit of the present assay is about 30 pM of the target DNA strand.     

Isolation of amaranthin synthetase from Chenopodium quinoa and construction of an amaranthin production system using suspension‐cultured tobacco BY‐2 cells

Betalains are plant pigments primarily produced by plants of the order Caryophyllales. Because betalain possesses anti‐inflammatory and anticancer activities, it may be useful as a pharmaceutical agent and dietary supplement. Recent studies have identified the genes involved in the betalain biosynthesis of betanin. Amaranthin and celosianin II are abundant in the quinoa (Chenopodium quinoa Willd.) hypocotyl, and amaranthin comprises glucuronic acid bound to betanin; therefore, this suggests the existence of a glucuronyltransferase involved in the synthesis of amaranthin in the quinoa hypocotyl. To identify the gene involved in amaranthin biosynthesis, we performed a BLAST analysis and phylogenetic tree analysis based on sequences homologous to flavonoid glycosyltransferase, followed by expression analysis on the quinoa hypocotyl to obtain three candidate proteins. Production of amaranthin in a transient Nicotiana benthamiana expression system was evaluated for these candidates and one was identified as having the ability to produce amaranthin. The gene encoding this protein was quinoa amaranthin synthetase 1 (CqAmaSy1). We also created a transgenic tobacco bright yellow‐2 (BY‐2) cell line wherein four betalain biosynthesis genes were introduced to facilitate amaranthin production. This transgenic cell line produced 13.67 ± 4.13 μm (mean ± SEM) amaranthin and 26.60 ± 1.53 μm betanin, whereas the production of isoamaranthin and isobetanin could not be detected. Tests confirmed the ability of amaranthin and betanin to slightly suppress cancer cell viability. Furthermore, amaranthin was shown to significantly inhibit HIV‐1 protease activity, whereas betanin did not.     

Fungal effector SIB1 of Colletotrichum orbiculare has unique structural features and can suppress plant immunity in Nicotiana benthamiana

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five β-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel β-barrel structure. However, the β-strands were found to display a unique topology, one pair of these β-strands formed a parallel β-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern–triggered immunity in N. benthamiana.     

L-Type Ca2+ Channel Sparklets Revealed by TIRF Microscopy in Mouse Urinary Bladder Smooth Muscle

Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca²⁺, referred to as Ca²⁺ sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca²⁺ imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca²⁺ (0.035±0.01 vs. 0.23±0.07 Hz/mm²; P<0.05). Co-loading of smooth muscle strips with the slow Ca²⁺ chelator EGTA-AM (10 mM) confirmed that Ca²⁺ sparklets are restricted to the cell membrane. Ca²⁺ sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 μM) (0.034±0.02 vs. 0.21±0.08 Hz/mm²; P<0.05), and diltiazem (10 μM) (0.097±0.04 vs. 0.16±0.06 Hz/mm²; P<0.05). Ca²⁺ sparklets were unaffected by inhibition of P2X₁ receptors α,β-meATP (10 μM) whilst sparklet frequencies were significantly reduced by atropine (1 μM). Ca²⁺ sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) (0.030±0.01 vs. 0.30±0.1 Hz/mm²; P<0.05), demonstrating that Ca²⁺ sparklets are PKC dependant. In the presence of CPA (10 μM), there was no apparent change in the overall frequency of Ca²⁺ sparklets, although the sparklet frequencies of each UBSM became statistically independent of each other (Spearman''s rank correlation 0.2, P>0.05), implying that Ca²⁺ store mediated signals regulate Ca²⁺ sparklets. Under control conditions, inhibition of store operated Ca²⁺ entry using ML-9 (100 μM) had no significant effect. Amplitudes of Ca²⁺ sparklets were unaffected by any agonists or antagonists, suggesting that these signals are quantal events arising from activation of a single channel, or complex of channels. The effects of CPA and ML-9 suggest that Ca²⁺ sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca²⁺ concentrations.     

Thermal membrane potential across charged membranes in NaCl-NH4Cl and LiCl-NH4Cl solutions

Measurements of the thermal membrane potential across cation exchange membranes were carried out by using aqueous solutions containing two 1-1 electrolytes, with an anion in common. The same solution was used on both sides of the membrane. In all cases a good linear relationship was observed between the thermal membrane potential Δψ and the temperature difference ΔT (in the range ΔT = ± 10°C). Assuming that the activity of one cation is equal to that of another cation in the solutions and the sum of transport numbers of cations is unity, the plot of Δψ/ΔT vs logarithmic activity of one cation is linear with a slope of R/F. These experimental results aie in agreement with a theory presented previously. From the analysis of thermal membrane potential in mixtures of electrolytes it is obtained that the cross coefficient of cation-cation interaction in membranes is negative and about 6 to 9% of the main coefficient.