A high-potential nonheme iron protein (HiPIP)-linked,thiosulfate-oxidizing enzyme derived fromChromatium vinosum

A thiosulfate-oxidizing enzyme was partially purified fromChromatium vinosum, and some of its properties were studied. The enzyme rapidly reducede HiPIP (high-potential nonheme iron protein) in the presence of thiosulfate. Cytochromesc of yeast and tuna and ferricyanide also acted well as electron acceptors for the enzyme; horse cytochromec was a poor electron acceptor. Cytochromec-552, cytochromec′, and cytochromec-553 did not act as electron acceptors. The enzyme was inhibited by cyanide and sulfite. On the basis of the stoichiometry in reduction of ferricyanide catalyzed by the enzyme in the presence of thiosulfate, the oxidized product of thiosulfate was inferred to be tetrathionate.     

Novel mannose-binding rice lectin composed of some isolectins and its relation to a stress-inducible salT gene

The novel mannose-binding rice lectin (MRL) purified by Sephadex G-50 or maltamyl Sepharose 4B affinity chromatography was not homogeneous, but the components were separated clearly by two dimensional polyacrylamide gel electrophoresis (1st; isoelectric focusing with Immobiline, 2nd; SDS-PAGE). The major spots were located at pI 4.85 and 4.74, and minor spots at pI 4.66, 4.56, and 4.44; all spots were distributed at about MW 45,000. Other faint spots were sometimes detected just below the major spots. In the western blot analysis, all the spots reacted with the monoclonal antibodies specific to MRL, which bound to MRL and inhibited the lectin activity to agglutinate rabbit erythrocytes. The proteins of the spots at pI 4.85, 4.77, 4.66, and 4.56 had lectin activity. The major proteins at pI 4.85 and 4.77 also had the common amino acid sequence at N-terminus, TLVKIGPWGGNGGSAQDISV, which is almost identical to salt and drought stress-inducible salT gene products in rice plants. High homology was also conserved in both the cDNA and the genomic clones encoding the MRL component at pI 4.85, which were selected with MRL-specific antibodies and an oligonucleotide designed from the partial amino acid sequence. All results suggest that MRL is composed of several isolectins, if not, related proteins having a common epitope and may belong to a family of stress-inducible proteins.     

Environmental DNA monitoring method of the commercially important and endangered fish Gnathopogon caerulescens

Limnology - Gnathopogon caerulescens is an endangered but commercially important fish in Lake Biwa, Japan. The population size of G. caerulescens has drastically reduced in the past decades, and...     

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.     

Stem cells assessed

The increasing momentum of stem cell research continues, with the better characterization of induced pluripotent stem (iPS) cells, the conversion of differentiated cells into different cell types and the use of pluripotent stem cells to generate whole tissues, among other advances. Here, six experts in the field of stem cell research compare different stem cell models and highlight the importance of pursuing complementary experimental approaches for a better understanding of pluripotency and differentiation and an informed approach to medical applications.     

Detection of β-glucosidase as saprotrophic ability from an ectomycorrhizal mushroom, Tricholoma matsutake

We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects. We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized. The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca²⁺ and Mn²⁺ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction.     

Newly established in vitro system with fluorescent proteins shows that abnormal expression of downstream prion protein-like protein in mice is probably due to functional disconnection between splicing and 3' formation of prion protein pre-mRNA

We and others previously showed that, in some lines of prion protein (PrP)-knockout mice, the downstream PrP-like protein (PrPLP/Dpl) was abnormally expressed in brains partly due to impaired cleavage/polyadenylation of the residual PrP promoter-driven pre-mRNA despite the presence of a poly(A) signal. In this study, we newly established an in vitro transient transfection system in which abnormal expression of PrPLP/Dpl can be visualized by expression of the green fluorescence protein, EGFP, in cultured cells. No EGFP was detected in cells transfected by a vector carrying a PrP genomic fragment including the region targeted in the knockout mice intact upstream of the PrPLP/Dpl gene. In contrast, deletion of the targeted region from the vector caused expression of EGFP. By employing this system with other vectors carrying various deletions or point mutations in the targeted region, we identified that disruption of the splicing elements in the PrP terminal intron caused the expression of EGFP. Recent lines of evidence indicate that terminal intron splicing and cleavage/polyadenylation of pre-mRNA are functionally linked to each other. Taken together, our newly established system shows that the abnormal expression of PrPLP/Dpl in PrP-knockout mice caused by the impaired cleavage/polyadenylation of the PrP promoter-driven pre-mRNA is due to the functional dissociation between the pre-mRNA machineries, in particular those of cleavage/polyadenylation and splicing. Our newly established in vitro system, in which the functional dissociation between the pre-mRNA machineries can be visualized by EGFP green fluorescence, may be useful for studies of the functional connection of pre-mRNA machineries.