Non-genomic effects of aldosterone on intracellular ion regulation and cell volume in rat ventricular myocytes

Aldosterone has non-genomic effects that express within minutes and modulate intracellular ion milieu and cellular function. However, it is still undefined whether aldosterone actually alters intracellular ion concentrations or cellular contractility. To clarify the non-genomic effects of aldosterone, we measured [Na+]i, Ca2+ transient (CaT), and cell volume in dye-loaded rat ventricular myocytes, and we also evaluated myocardial contractility. We found the following: (i) aldosterone increased [Na+]i at the concentrations of 100 nmol/L to 10 micromol/L; (ii) aldosterone (up to 10 micromol/L) did not alter CaT and cell shortening in isolated myocytes, developed tension in papillary muscles, or left ventricular developed pressure in Langendorff-perfused hearts; (iii) aldosterone (100 nmol/L) increased the cell volume from 47.5 +/- 3.6 pL to 49.8 +/- 3.7 pL (n=8, p<0.05); (iv) both the increases in [Na+]i and cell volume were blocked by a Na+-K+-2Cl- co-transporter (NKCCl) inhibitor, bumetanide, or by a Na+/H+ exchange (NHE) inhibitor, 5-(N-ethyl-N-isopropyl) amiloride; and (v) spironolactone by itself increased in [Na+]i and cell volume. In conclusion, aldosterone rapidly increased [Na+]i and cell volume via NKCC1 and NHE, whereas there were no changes in CaT or myocardial contractility. Hence the non-genomic effects of aldosterone may be related to cell swelling rather than the increase in contractility.     

alpha-Tomatine, the major saponin in tomato, induces programmed cell death mediated by reactive oxygen species in the fungal pathogen Fusarium oxysporum

The tomato saponin alpha-tomatine has been proposed to kill sensitive cells by binding to cell membranes followed by leakage of cell components. However, details of the modes of action of the compound on fungal cells are poorly understood. In the present study, mechanisms involved in alpha-tomatine-induced cell death of fungi were examined using a filamentous pathogenic fungus Fusarium oxysporum. alpha-Tomatine-induced cell death of F. oxysporum (TICDF) occurred only under aerobic conditions and was blocked by the mitochondrial F(0)F(1)-ATPase inhibitor oligomycin, the caspase inhibitor D-VAD-fmk, and protein synthesis inhibitor cycloheximide. Fungal cells exposed to alpha-tomatine showed TUNEL-positive nuclei, depolarization of transmembrane potential of mitochondria, and reactive oxygen species (ROS) accumulation. These results suggest that TICDF occurs through a programmed cell death process in which mitochondria play a pivotal role. Pharmacological studies using inhibitors suggest that alpha-tomatine activates phosphotyrosine kinase and monomeric G-protein signaling pathways leading to Ca(2+) elevation and ROS burst in F. oxysporum cells.     

Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.     

Proteomic and microarray analyses of the Dictyostelium Zak1-GSK-3 signaling pathway reveal a role in early development

GskA, the Dictyostelium GSK-3 orthologue, is modified and activated by the dual-specificity tyrosine kinase Zak1, and the two kinases form part of a signaling pathway that responds to extracellular cyclic AMP. We identify potential cellular effectors for the two kinases by analyzing the corresponding null mutants. There are proteins and mRNAs that are altered in abundance in only one or the other of the two mutants, indicating that each kinase has some unique functions. However, proteomic and microarray analyses identified a number of proteins and genes, respectively, that are similarly misregulated in both mutant strains. The positive correlation between the array data and the proteomic data is consistent with the Zak1-GskA signaling pathway's functioning by directly or indirectly regulating gene expression. The discoidin 1 genes are positively regulated by the pathway, while the abundance of the H5 protein is negatively regulated. Two of the targets, H5 and discoidin 1, are well-characterized markers for early development, indicating that the Zak1-GskA pathway plays a role in development earlier than previously observed.     

Chromosome-Scale Genome Assembly of the Marine Oleaginous Diatom Fistulifera solaris

Marine Biotechnology - Microalgae including diatoms are of interest for environmentally friendly manufacturing such as production of biofuels, chemicals, and materials. The highly oil-accumulating...     

Production and characterization of recombinant P1 adhesin essential for adhesion,gliding, and antigenic variation in the human pathogenic bacterium,Mycoplasma pneumoniae

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.     

Koji glycosylceramide commonly contained in Japanese traditional fermented foods alters cholesterol metabolism in obese mice

ABSTRACT

Koji, which is manufactured by proliferating non-pathogenic fungus Aspergillus oryzae on steamed rice, is the base for Japanese traditional fermented foods. We have revealed that koji and related Japanese fermented foods and drinks such as amazake, shio-koji, unfiltered sake and miso contain abundant glycosylceramide. Here, we report that feeding of koji glycosylceramide to obese mice alters the cholesterol metabolism . Liver cholesterol was significantly decreased in obese mice fed with koji glycosylceramide. We hypothesized that their liver cholesterol was decreased because it was converted to bile acids. Consistent with the hypothesis, many bile acids were increased in the cecum and feces of obese mice fed with koji glycosylceramide. Expressions of CYP7A1 and ABCG8 involved in the metabolism of cholesterol were significantly increased in the liver of mice fed with koji glycosylceramide. Therefore, it was considered that koji glycosylceramide affects the cholesterol metabolism in obese mice.     

Separation and properties of two acetylacetoin reductases from Bacillus cereus YUF-4

The separation and purification of two kinds of acetylacetoin reductases (AACRs) from Bacillus cereus YUF-4 were examined. NADPH-linked AACR (AACR I) and NADH-linked AACR (AACR II) were separated from each other by ammonium sulfate fractionation, DEAE-cellulose chromatography, and hydroxyapatite chromatography. The former was purified 3.4-fold with a yield of 10.0%, and the latter was purified 29-fold with a yield of 15.6%. The two enzymes differ from each other in some enzymic properties such as substrate specificity.