Measurement of human urokinase activity in plasma by using mono-specific antibody-conjugated paper disk

Human urokinase (HUK) was purified from commercial product by high performance liquid chromatography on TSK GEL-G3000SW and affinity chromatography on benzamidine-Sepharose 4B. The purified enzyme was of a high molecular weight form (molecular weight of 53,000). This preparation was utilized as an antigen to immunize rabbits; the obtained antibody showed a high specificity against HUK. The antibody was conjugated to CNBr-activated paper disks. The antibody-conjugated paper disk and a fluorogenic peptide substrate, glutaryl-Gly-Arg-4-methyl-coumarine-7-amide, were used to measure urokinase (UK) activity in plasma. The calibration curve obtained by the proposed method passed through the origin and was linear in the range of 0-0.16 IU of HUK. The incubation of HUK with an excess amount of alpha 2-macroglobulin at 37 degrees C for 3 h gave only about a 30% decrease of the activity assayed by the proposed method. After incubations of HUK with alpha 1-antitrypsin and antithrombin III, the activity was completely inhibited. The incubation of HUK with plasma at 37 degrees C decreased the activity as a function of time. However, when the antibody-conjugated paper disk was used for the immunoreaction to HUK in plasma at 4 degrees C, no decrease of UK activity was observed. The plasma decay curve of UK activity after a single intravenous (i.v.) injection of HUK into a rabbit (12,000 IU/kg) indicated bi-exponential kinetics by using this assay method. The rate constants of the alpha and beta phases were 0.120 +/- 0.020 and 0.021 +/- 0.002 min-1, respectively. These result suggest that the proposed method is useful for measuring UK activity in plasma of patients with intravascular coagulation after i.v. administration of UK.     

Micropropagation of horseradish hairy root by means of adventitious shoot primordia

Adventitious shoot primordia were formed on horseradish hairy root cultured in dark. Plantlet formation frequency from the primordia was higher than that from root fragments. Culture for 26 days provided the adventitious shoot primordia, which had the highest potential for plantlet formation (53% explants at 40 days). Benzyladenine supplementation in the dark caused primordium enlargement, but did not increase the number of primordia formed. After adventitious shoot primordia were encapsulated with calcium alginate, kinetin supplementation (2.0–4.0 M) increased the shoot formation frequency (65–80% explants at 20 days) in the light, but also promoted the undesirable formattion of multiple shoots. Supplementation with naphthaleneacetic acid (0.27–5.4 M) in the calcium alginate beads in light enhanced the root emergence from primordia without inhibition of plantlet formation when the encapsulated beads were put on the agar-medium without naphthaleneacetic acid.     

Glycosphingolipids in cultured lens epithelial cells from dog and rhesus monkey

Vertebrate lens tissues contain several species of acidic andneutral glycosphingolipids in relatively high amounts. However,the epithelia with capsule from dog and rhesus monkey lenseshad a simpler composition and lower content of glycosphingolipidsthan whole lenses. Gangliosides and neutral glycosphingolipidsin monolayer cultures of lens epithelial cells were also differentfrom those in whole lenses. Although -galactosyl (Gal1-3Ga1-R)or Lewis^x (Galß1-4[Fuc1-3]GlcNAc-R) epitopes werefound in glycosphingolipids from whole lenses, they were notdetected in those from monolayer cultures of dog and rhesusmonkey lens cells. In addition, significant changes in ganglio-seriesgangliosides were induced in monolayer cultures of both cells,where GM3 and GD3 were predominant. Immunofluorescence studyrevealed a characteristic distribution of cell surface gangliosidesin confluent monolayers. These findings suggest that glycosphingolipidsynthesis in lens epithelia is intrinsically different fromthat in cortical and nuclear fibres, and that the expressionof Lewis^x and -galactosyl epitopes in glycosphingolipids appearsto be associated with the differentiation of epithelial cellsto fibres. gangliosides glycosphingolipids lens epithelial cells Lewis^x rhesus monkey.     

The first report of the non-indigenous Chydorus brevilabris Frey, 1980 (Crustacea: Cladocera) in Asian freshwaters

Limnology - Chydorus sphaericus (O.F. Müller, 1776) (Crustacea: Cladocera) sensu stricto is distributed in Europe: C. sphaericus-like organisms in other regions represent a group of...     

A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene

Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.     

Neural Substrates for Judgment of Self-Agency in Ambiguous Situations

The sense of agency is the attribution of oneself as the cause of one’s own actions and their effects. Accurate agency judgments are essential for adaptive behaviors in dynamic environments, especially in conditions of uncertainty. However, it is unclear how agency judgments are made in ambiguous situations where self-agency and non-self-agency are both possible. Agency attribution is thus thought to require higher-order neurocognitive processes that integrate several possibilities. Furthermore, neural activity specific to self-attribution, as compared with non-self-attribution, may reflect higher-order critical operations that contribute to constructions of self-consciousness. Based on these assumptions, the present study focused on agency judgments under ambiguous conditions and examined the neural correlates of this operation with functional magnetic resonance imaging. Participants performed a simple but demanding agency-judgment task, which required them to report on whether they attributed their own action as the cause of a visual stimulus change. The temporal discrepancy between the participant’s action and the visual events was adaptively set to be maximally ambiguous for each individual on a trial-by-trial basis. Comparison with results for a control condition revealed that the judgment of agency was associated with activity in lateral temporo-parietal areas, medial frontal areas, the dorsolateral prefrontal area, and frontal operculum/insula regions. However, most of these areas did not differentiate between self- and non-self-attribution. Instead, self-attribution was associated with activity in posterior midline areas, including the precuneus and posterior cingulate cortex. These results suggest that deliberate self-attribution of an external event is principally associated with activity in posterior midline structures, which is imperative for self-consciousness.     

Spatial heterogeneity in induced defense of Brachionus calyciflorus within a single lake caused by a bed of floating-leaved macrophyte Trapa species

Limnology - While induced defense of aquatic organisms against predators has been considerably studied by both laboratory and field research, our understanding is still limited about, for example,...     

Transcriptome analysis of soybean (Glycine max) root genes differentially expressed in rhizobial,arbuscular mycorrhizal,and dual symbiosis

Journal of Plant Research - Soybean (Glycine max) roots establish associations with nodule-inducing rhizobia and arbuscular mycorrhizal (AM) fungi. Both rhizobia and AM fungi have been shown to...