Guinea pig anaphylaxis: time course of changes in cyclic AMP and cyclic GMP.

The time course of changes in the levels of cyclic AMP and GMP and in histamine and prostaglandin release following the onset of anaphylaxis in guinea pig lung (sliced, minced and whole perfused) was studied. A parallel change in the cyclic nucleotide levels was found. There was an initial increase, followed by a decrease, and a subsequent greater and more prolonged increase. Significant histamine and prostaglandin release occurs at approximately the same time while prostaglandin metabolite is released later. Metiamide does not affect histamine or prostaglandin release but abolishes the initial cyclic nucleotide peak and delays the appearance of the secondary increase. The pattern of cyclic nucleotide changes in lung tissue thus appears to be different from that in isolated mast cells. The initial and late increases might be the response of two different cell populations to histamine.     

Encapsulation of whole cell CGTase from concentrated broth solution

Most of the Cyclodextrin glucanotransferase (Gtases) which have been produced fromB. subtilis were found to be excreted from the cells during cultivation. Immobilized whole cell CGTase fromB. subtilis was prepared by encapsulating the broth solution which had been concentrated ten times with a rotary vacuum evaporator. Cyclization activity of CGTase was reduced by about 10% during the concentrating process, however, its transglycosylation activity, to convert xylitol to glucosyl-xylitol, using dextrin as glucosyl donor, increased by a factor of 3 or 5.     

Fishborne Trematode Metacercariae Detected in Freshwater Fish from Vientiane Municipality and Savannakhet Province, Lao PDR

Freshwater fish from Vientiane Municipality and Savannakhet Province, Lao PDR were examined by the muscle compression and artificial digestion methods to know the infection status with trematode metacercariae. In the fish from Savannakhet, 2 species of metacercariae, Opisthorchis viverrini and Haplorchis taichui, were detected. O. viverrini metacercariae were found in 6 species of fish, Puntius brevis, Hampala dispar, Esomus metallicus, Mystacoleucus marginatus, Puntioplites falcifer, and Cyclocheilichthys armatus. H. taichui metacercariae were detected in 3 species of fish, P. brevis, P. falcifer, and M. marginatus. In the fish from Vientiane, 4 species of metacercariae, O. viverrini, H. taichui, Haplorchis yokogawai, and Centrocestus formosanus, were detected. Among them, O. viverrini metacercariae were found in 7 species of fish, Onychostoma elongatum, C. armatus, H. dispar, P. brevis, Cyclocheilichthys repasson, Osteochilus hasseltii, and Hypsibarbus lagleri. The metacercariae of H. taichui were detected in 6 species of fish, C. repasson, O. elongatum, C. armatus, H. dispar, Labiobarbus leptocheila, and Cirrhinus molitorella. The metacercariae of H. yokogawai were found in 9 species of fish, C. repasson, O. elongatum, C. armatus, H. dispar, Labiobarbus leptocheila, O. hasseltii. C. molitorella, Hypsibarbus wetmorei, and H. lagleri. The metacercariae of C. formosanus were detected in 4 species of fish, C. repasson, P. brevis, O. hasseltii, and C. molitorella. From these results, it is confirmed that fishborne trematode metacercariae, i.e. O. viverrini, H. taichui, H. yokogawai and C. formosanus, are prevalent in various species of freshwater fish from Savannakhet Province and Vientiane Municipality, Lao PDR.     

Vitellocyte-specific expression of phospholipid hydroperoxide glutathione peroxidases in Clonorchis sinensis

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and may protect against lipid hydroperoxidation in biomembranes. We isolated full-length cDNA sequences encoding four different PHGPxs from a causative agent of cholangiocarcinoma, Clonorchis sinensis (CsGPx1, CsGPx2, CsGPx3 and CsGPx4). These sequences contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in their 3′-untranslated regions. The open reading frames were composed of six exons in the chromosomal segments of CsGPx1 (7705 bp), CsGPx2 (5871 bp) and CsGPx3 (3867 bp) and five exons in CsGPx4 (5655 bp). The positions of these introns were tightly conserved between the trematode and vertebrate PHGPx genes. Oxidative stimulation of viable worms with H₂O₂ or paraquat resulted in 1.5- to 2-fold induction of the GPx activity. The CsGPx proteins were specifically localised in vitellocytes within vitelline follicles and premature eggs in the proximal uterus. In the eggs, glutathione, an electron donor for GPx, was co-localised with the CsGPx proteins, while thioredoxin, which is preferred by peroxiredoxin, was principally detected in the extracellular space between the embryonic cell mass and an eggshell. Our data may suggest a concerted or a specialised function between a thioredoxin-dependent enzyme(s) and GPx in protecting against H₂O₂-derived damage during maturation of the embryo and formation of the eggshell, in these catalase-lacking trematode parasites. The uniquely conserved genomic organisation and Sec-dependency amongst trematode and vertebrate PHGPx homologues will also provide insight into the evolutionary episode and functional/biochemical diversification of GPx proteins.     

Brain cancer stem-like cell genesis from p53-deficient mouse astrocytes by oncogenic Ras

Here, we show that H-ras^V12 causes the p53-knockout mouse astrocytes (p53−/− astrocytes) to be transformed into brain cancer stem-like cells. H-ras^V12 triggers the p53−/− astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras^V12 also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras^V12-overexpressing p53−/− astrocytes (p53−/−ast-H-ras^V12) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.     

Anti-oxidant effect of ascorbic and dehydroascorbic acids in hippocampal slice culture

Ascorbic acid (AA) and dehydroascorbic acid (DHA) have been shown to have protective effects as anti-oxidants in experimental neurological disorder models such as stroke, ischemia, and epileptic seizures. The present study was conducted to examine the protective effects of AA and DHA on kainic acid (KA) neurotoxicity using organotypic hippocampal slice cultures. After 12 h KA treatment, significant delayed neuronal death was detected in the CA3, but not the CA1, region. Pretreatment with intermediate doses of AA and DHA significantly prevented cell death and inhibited reactive oxygen species (ROS) level, and mitochondrial dysfunction in the CA3 region. In contrast, pretreatment with low or high doses of AA or DHA was not effective. These data suggest that pretreatment with both AA and DHA has dose-dependent neuroprotective effects on KA-induced neuronal injury through inhibiting ROS generation and mitochondrial dysfunction.     

Molecular cloning of stanniocalcin 1 and its extracorpuscular regulation by salinity and Ca2+ in the Japanese flounder

Stanniocalcin 1 (Stc1) was originally identified as an anti-hypercalcemic hormone produced by the corpuscles of Stannius (CS) associated with the kidney in teleosts. While the stc1 gene is expressed in various tissues in fishes, its role and regulation in extra-CS tissues are unexplored. In the present study, we characterized a cDNA of stc1 in a euryhaline fish, the Japanese flounder (Paralichyhus olivaceus), and examined its expression in peripheral tissues in response to different salinities and Ca2+ ion concentrations. The Japanese flounder stc1 cDNA (1331 bp) encodes a preprohormone of 251 amino acids (aa), with a signal peptide of 17 aa and a pro-sequence peptide of 15 aa followed by the mature protein of 219 aa. The deduced aa sequence of Japanese flounder stc1 showed highest sequence identity (94.0%) with the European flounder Stc1 among fish and mammalian species, but lower identity to zebrafish, pufferfish, and human STC2 (23.1-25.4%). Lowered environmental salinity resulted in a decrease in stc1 mRNA expression in vivo in the gills, kidney, intestine, and CS glands of the Japanese flounder. Furthermore, we found that extracellular Ca2+ increased steady-state stc1 mRNA levels in gill and kidney cells as well as in the CS cells. Our findings suggest that Stc1 synthesis in the ionregulatory tissues is responsive to environmental salinity and Ca2+ level.     

Algicidal activity of the seaweed Corallina pilulifera against red tide microalgae

Extracts of seaweeds from the coast of Korea have been tested in vitro for algicidal activity against the growth of the toxic microalga Cochlodinium polykrikoides. Blooms of C. polykrikoides and the ensuing mass mortalities of farmed fish and shellfish are an escalating and worrisome trend. Cell growth of C. polykrikoides was inhibited by the addition to the culture medium of several seaweed extracts. Inhibition of growth resulted from methanol-soluble extracts of the seaweeds Corallina pilulifera, Ulva pertusa, Ishige foliacea and Endarachne binghamiae. Growth inhibition also resulted from the water-soluble extract of C. pilulifera. Powder and dry tissue from the seaweed C. pilulifera also inhibited cell growth of C. polykrikoides. The active algicidal products of C. pilulifera showed stable activity when boiled, exposed to light, or when treated under alkaline condition. Corallina pilulifera had no regional and seasonal variations in this algicidal activity. A powder of the seaweed C. pilulifera, the most potent species, showed algicidal activity against several red tide microalgae, especially C. polykrikoides, Gymnodiniummikimotoi, G. sanguineum, Heterosigma akashiwo, Prorocentrum triestinum and Pyraminonas sp. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca²⁺-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α₁-antitrypsin (α₁-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α₁-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.