Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

ε Subunit of Bacillus subtilis F1-ATPase Relieves MgADP Inhibition

MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F₁-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F₁-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F₁-ATPase from Bacillus subtilis (BF₁), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF₁. We conclude that the ε subunit relieves BF₁ from MgADP inhibition.     

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.     

Anti-cancer effects of naturally occurring compounds through modulation of signal transduction and miRNA expression in human colon cancer cells

Much evidence indicates that various naturally occurring compounds have an anti-cancer effect, but the detailed mechanisms are not well understood. In this study, we selected anti-cancer phytochemicals such as epigallocatechin-3-gallate (EGCG), resveratrol (RES) and α-mangostin (α-M), all of which are well-characterized chemopreventive agents. We sought to elucidate the mechanism of their anti-cancer effects and the synergistic effects obtained by combined treatment with the anti-cancer drug 5-fluorouracil (5-FU) in three human colon cancer cell lines. The numbers of viable cells were consistently decreased by the treatment with EGCG, RES or α-M at more than 10 μM in all three cell lines tested. All compounds mainly induced apoptosis and suppressed the PI3K/Akt signaling pathway. Additionally, α-M, which had the greatest PI3K/Akt-suppressing activity, also suppressed MAP kinase (MAPK)/Erk1/2 signaling. Importantly, the combination treatment with RES and 5-FU induced a remarkably synergistic enhancement of growth inhibition and apoptosis through the additional suppression of the MAPK/Erk1/2 signaling pathway in colon cancer DLD-1 cells. Interestingly, RES increased the intracellular expression level of miR-34a, which down-regulated the target gene E2F3 and its downstream Sirt1, resulting in growth inhibition. These findings indicate that these compounds functioned as chemosensitizers when combined with anti-cancer drugs through the modulation of apoptotic and growth-related signaling pathways. Also, RES exerted its anti-cancer activity in part through a newly defined mechanism, i.e., the miR-34a/E2F3/Sirt1 cascade.     

Acid-Catalyzed Stereoselective Cyclization of Germacrene D

A monohalomethane-producing enzyme, S-adenosyl-L-methionine-dependent halide ion methyltransferase (EC 2.1.1.-) was purified from the marine microalga Pavlova pinguis by two anion exchange, hydroxyapatite and gel filtration chromatographies. The methyltransferase was a monomeric molecule having a molecular weight of 29,000. The enzyme had an isoelectric point at 5.3, and was optimally active at pH 8.0. The K_m for iodide and SAM were 12 mM and 12 μM, respectively, which were measured using a partially purified enzyme. Various metal ions had no significant effect on methyl iodide production, suggesting that the enzyme does not require metal ions. The enzyme reaction strictly depended on SAM as a methyl donor, and the enzyme catalyzed methylation of the I^-,Br^-, and Cl^- to corresponding monohalomethanes and of bisulfide to methyl mercaptan.     

Studies on the Degradation Mechanisms of Proteins and their Derivatives in the Foods

The volatile sulfur components produced by boiling soybean meal hydrolyzates (AMINOSAN-EKI) have been identified as dimethyl sulfide and hydrogen sulfide. No mercaptan or disulfides were detected.

The main precursor of dimethyl sulfide is supposed to be methionine methylsulfonium compound derived from methionine and pectin substances (–COOCH₃) during the hydrolysis of soybean meal by hydrochloric acid.     

New Coumaronochromones from White Lupin,Lupinus albus L. (Leguminosae)

A further investigation of the isoflavonoid constituents occurring in roots of the white lupin (Lupinus albus L. cv. Kievskij Mutant) has yielded five new coumaronochromones named lupinalbin A (la), B (2a), C (3), D (4) and E (5). These isoflavonoids were identified by physicochemical methods involving the use of biogenetically related 2′-hydroxyisoflavones as reference compounds. The presence of the rare dihydrofurano-isoflavone, erythrinin C (16), in white lupin roots has also been established.     

Studies on the Degradation Mechanisms of Proteins and their Derivatives in the Foods

Cysteine-aldehyde compounds were prepared by the reactions of l-cysteine with formaldehyde, acetaldehyde, n-butyraldehyde, benzaldehyde and furfural in 50% ethanol solutions. Hydrogen sulfide and ammonia liberated from cysteine-aldehyde compounds in heated aqueous solutions (oil bath : 120°C) were determined. Although thiazolidine derivatives were stable generally in boiling aqueous solution, l-cysteine-furfural compound was unstable and a large amount of hydrogen sulfide compared with other compounds was released.