Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxii

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.     

Differential desensitization and internalization of three different bullfrog gonadotropin-releasing hormone receptors

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.     

Interleukin-4 inhibits the vascular endothelial growth factor- and basic fibroblast growth factor-induced angiogenesis in vitro

The role of interleukin-4 (IL-4) in the inflammatory process has emerged recently. In this study, we investigated the effect of IL-4 on the angiogenic process in an in vitro experimental system. IL-4 significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVEC) that was induced by the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF- or bFGF-induced HUVEC chemotaxis was abrogated by the IL-4 treatment. In addition, the formation of tube-like structures by HUVEC in the presence of VEGF or bFGF was also severely down-regulated by IL-4. The inhibitory effects on the critical steps of angiogenesis were not observed with IL-6 that is abundantly found in the inflamed tissue. Our results suggest that IL-4 may play a regulatory role in normal physiology and provide the potential possibility for IL-4 as a therapeutic agent in the intervention of angiogenesis-related diseases.     

Klebsiella pneumoniae KE-1 degrades endosulfan without formation of the toxic metabolite,endosulfan sulfate

For bioremediation of toxic endosulfan, endosulfan degradation bacteria, which do not form toxic endosulfan sulfate, were isolated from various soil samples using endosulfan as sole carbon and energy source. Among the 40 isolated bacteria, strain KE-1, which was identified as Klebsiella pneumoniae by physiological and 16S rDNA sequence analysis, showed superior endosulfan degradation activity. Analysis of culture pH, growth, free sulfate and endosulfan and its metabolites demonstrated that KE-1 biologically degrades 8.72 microg endosulfan ml(-1) day(-1) when incubated with 93.9 microg ml(-1) endosulfan for 10 days without formation of toxic endosulfan sulfate. Our results suggest that K. pneumoniae KE-1 degraded endosulfan by a non-oxidative pathway and that strain KE-1 has potential as a biocatalyst for endosulfan bioremediation.     

EGCG attenuates AMPA-induced intracellular calcium increase in hippocampal neurons

We have investigated the protective effect of (-)-epigallocatechin gallate (EGCG) on alpha-amino-3-hydroxy-5-methyl-4-isoxazolo propionate (AMPA)-induced toxicity in cultured rat hippocampal neurons. Treatment of 24 h AMPA (10 microM) reduced the neuronal viability in both survival neuron counting and MTT reduction assay compared with control, with increase in cellular concentrations of hydrogen peroxide and malondialdehyde. These responses to AMPA were significantly blocked by co-treatments with EGCG (10 microM), which effect was very similar to the protective ability of a known antioxidant catalase (2000 U/ml). AMPA (50 microM) elicited the increase in intracellular calcium concentration ([Ca(2+)]i) on which EGCG significantly attenuated both peak amplitude and sustained nature of that [Ca(2+)]i increase in a dose-dependent manner. These data suggest that EGCG has a neuroprotective effect against AMPA through inhibition of AMPA-induced [Ca(2+)]i increase and consequent attenuation of reactive oxygen species production and lipid peroxidation as an antioxidant and a radical scavenger.     

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.     

Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.     

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.