Use of electrophoretic enzyme patterns for streptomycete systematics

Abstract The relative electrophoretic mobilities of various enzymes from 24 different streptomycetes were determined on polyacrylamide gels in order to examine the relatedness of species and strains of the genus Streptomyces . Of 11 different enzymes tested in this study, hexokinase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase showed a limited number of constant and reproducible polymorphic enzyme patterns, by comparing which the inter-specific relationships could be examined. In contrast, glucose dehydrogenase, alcohol dehydrogenase, 3-hydroxybutyrate dehydrogenase, phosphoglucose isomerase, peroxidase and esterase exhibited either weak non-reproducible or highly heterogeneous band patterns which were suitable for dissecting the strains within a species and a cluster group.     

Syntheses of sphingosine-1-phosphate stereoisomers and analogues and their interaction with EDG receptors

Sphingosine-1-phosphate (S1P) is considered to be an important regulator of diverse biological processes acting as a natural ligand to EDG receptors. As a preliminary study to develop potent and selective agonist and antagonist for EDG receptors, we report synthesis of S1P stereoisomers and analogues and their binding affinities to EDG-1, -3, and -5.     

Abnormal growth of polyamine-deficient Escherichia coli mutant is partially caused by oxidative stress-induced damage

Polyamines participate in numerous cellular processes and are required for normal cell growth in Escherichia coli. In this study, we constructed a new polyamine-deficient E. coli mutant and investigated the physiological function of polyamines during normal aerobic growth conditions. We showed that the requirement for sulfur-containing, branched chain, and aromatic amino acids, which was exhibited in the sodA sodB double mutant faced with severe oxidative stress, was also true of the polyamine-deficient mutant during normal aerobic cell growth. Sorbitol, sucrose, mannose, 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an antioxidant that functions as an oxygen radical scavenger including z.rad;O(2)(-), and thiamine partially relieved the cell growth defect caused by polyamine depletion in a dose-dependent manner. As was the case for the cells treated with paraquat, the mutant had an elongated shape compared with the polyamine-proficient wild type. Decreased aeration also relieved the cell growth defect of the polyamine-deficient mutant. Finally, we confirmed that chloromethyl-2('),7(')-dichlorofluorescin diacetate (DCFH-DA), which is oxidized in a fluorescent product in the presence of various oxidants, also fluoresce in the polyamine-deficient cells. These results showed that abnormal growth of the polyamine-deficient E. coli mutant results partially from oxidative stress-induced damage and the mutant thus exhibits the requirement for antioxidant or specific nutritional amino acid during normal aerobic growth.     

The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine

In diagnostic pathology and immunocytochemical research, immunohistochemical techniques using the streptavidin–biotin–peroxidase system have played an extremely valuable role. This system, based on the high affinity of streptavidin for biotin, may, however, provoke false positive results because of endogenous streptavidin-binding sites in human tissues. With the advent of the antigen retrieval procedure and signal amplification method, this problem can be serious enough to cause mistakes in interpreting immunohistochemical staining results. Therefore, we examined the distribution of endogenous biotin-like molecules in various human tissues and the influence of various antigen retrieval procedures with or without signal amplification using biotinylated tyramine to reveal these biotin-like activities. We observed that endogenous biotin-like molecules were present in a wide range of tissues, and their activity was markedly enhanced by employing antigen retrieval procedures or signal amplification. Furthermore, the extent to which the activity of endogenous biotin-like activities was enhanced depended on the kinds of antigen retrieval procedures and signal amplification employed. Pressure cooking and tyramine amplification with microwave heating showed the highest activities. These results show that the antigen retrieval procedures and signal amplification with tyramine can enhance the activity of endogenous biotin or biotin-like molecules as well as antigenicity, which can be a pitfall in the interpretation of immunohistochemical data.     

IN VITRO ANTIOXIDANT ACTIVITIES OF THE FERMENTED MARINE MICROALGA PAVLOVA LUTHERI (HAPTOPHYTA) WITH THE YEAST HANSENULA POLYMORPHA1

Microalgae are major primary producers of organic matter in aquatic environments through their photosynthetic activities. Fermented microalga (Pavlova lutheri Butcher) preparation (FMP) is the product of yeast fermentation by Hansenula polymorpha. It was tested for the antioxidant activities including lipid peroxidation inhibitory activity, free‐radical‐scavenging activity, inhibition of reactive oxygen species (ROS) on mouse macrophages (RAW264.7 cell), and inhibited myeloperoxidase (MPO) activity in human myeloid cells (HL60). FMP exhibited the highest antioxidant activity on free‐radical scavenging, inhibitory intracellular ROS, and inhibited MPO activity. MTT [3‐(4,5‐dimethyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay showed no cytotoxicity in mouse macrophages (RAW264.7 cell), human myeloid cells (HL60), and human fetal lung fibroblast cell line (MRC‐5). Furthermore, the antioxidative mechanism of FMP was evaluated by protein expression levels of antioxidant enzyme (superoxide dismutase [SOD] and glutathione [GSH]) using Western blot. The results obtained in the present study indicated that FMP is a potential source of natural antioxidant.     

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6?. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine-bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.     

Expression of the tobacco mosaic virus movement protein alters starch accumulation inNicotiana benthamiana

Nicotiana benthamiana plants were transformed with the movement protein (MP) gene of tobacco mosaic virus (TMV), usingAgrobacterium-mediated transformation. Plants regenerated from the transformed cells accumulated 30-kDa MP and complemented the activity of TMV MP when infected with chimeric TMVs containing defective MR These transgenic plants displayed stunting, pale-green leaves, and starch accumulations, indicating that TMV MP altered the carbon partitioning for leaves involved in TMV cell-to-cell movement.     

Comparative Molecular Modeling Study of Arabidopsis NADPH-Dependent Thioredoxin Reductase and Its Hybrid Protein

2-Cys peroxiredoxins (Prxs) play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC) was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C) of thioredoxin reductase (TrxR) in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx) domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D)), which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD) simulations on AtNTRC and AtNTRA-(Trx-D) proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD) for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D) cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D) because of following reasons: i) unstable and unfavorable interaction of the linker region, ii) shifted Trx domain, and iii) different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.