Influence of simulated acid snow stress on leaf tissue of wintering herbaceous plants

Acid snow might be an environmental stress factor for wintering plants since acid precipitates are locally concentrated in snow and the period in which ice crystals are in contact with shoots might be longer than that of acid precipitates in rain. In this study, 'equilibrium' and 'prolonged' freezing tests with sulfuric acid, which simulate situations of temperature depression and chronic freezing at a subzero temperature with acid precipitate as acid snow stress, respectively, were carried out using leaf segments of cold-acclimated winter wheat. When leaf segments were frozen in the presence of sulfuric acid solution (pH 4.0, 3.0 or 2.0) by equilibrium freezing with ice seeding, the survival rate of leaf samples treated with sulfuric acid solution of pH 2.0 decreased markedly. Leaf samples after supercooling to -4 and -8 degrees C in the presence of sulfuric acid solution (pH 2.0) without ice seeding were less damaged. When leaf samples were subjected to prolonged freezing at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0), the survival rates of leaf samples exposed to sulfuric acid decreased more than those of leaf samples treated with water. On the other hand, leaf samples were less damaged by prolonged supercooling at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0). The results suggest that an acid condition (pH 2.0) in the process of extracellular freezing and/or thawing promotes freezing injury of wheat leaves.     

Strain difference in expression of the adult-type polycystic kidney disease gene, pcy, in the mouse

DBA/2FG-pcy and C57BL/6FG-pcy congenic strains were established by transferring the polycystic kidney disease gene, pcy, to DBA/2 and C57BL/6 mice. We carried out pathological and hematological examinations of these strains at 4, 8, 16 and 30 weeks of age. In DBA/2FG-pcy mice more than 8 weeks of age, macroscopic renal cysts were observed on the surface of both kidneys. Their kidneys weight was significantly greater than in DBA/2 mice at all ages examined. Microscopic renal cysts were evenly distributed at 4, 8 and 16 weeks of age. At 30 weeks of age, the kidneys were filled with numerous polycysts. In C57BL/6FG-pcy mice, no macroscopic renal cysts were found until the animals were 30 weeks old, and the weight of their kidneys was greater than in B6 mice of the same age. From 8 weeks of age on, a limited number of microscopic renal cysts was observed, and many renal cysts were found adjacent to the enlarged Bowman's capsules. With age, the red blood cell count and hematocrit level decreased while the platelet count increased in both strains, with greater changes occurring in DBA/2FG-pcy mice than in C57 BL/6FG-pcy mice. These findings demonstrate that polycystic kidney disease exhibits strain differences in animals with a DBA/2 and C57BL/6 background. Our results suggest that phenotypic expression of the pcy gene in the mouse depends on genetic background, and that variations in the severity of human polycystic kidney disease may be explained, at least in part, by individual differences in genetic background.     

24(S)‐hydroxycholesterol is actively eliminated from neuronal cells by ABCA1

High cholesterol turnover catalyzed by cholesterol 24‐hydroxylase is essential for neural functions, especially learning. Because 24(S)‐hydroxycholesterol (24‐OHC), produced by 24‐hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH‐SY5Y neuron‐like cells as a model, we examined whether 24‐OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24‐OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24‐OHC efflux was stimulated in the presence of high‐density lipoprotein (HDL), whereas apolipoprotein A‐I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24‐OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A‐I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24‐OHC. These results suggest that ABCA1 actively eliminates 24‐OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.     

G(i)-dependent activation of c-Jun N-terminal kinase in human embryonal kidney 293 cells

Heterotrimeric G proteins stimulate the activities of two stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase in mammalian cells. In this study, we examined whether alpha subunits of G(i) family activate JNK using transient expression system in human embryonal kidney 293 cells. Constitutively activated mutants of Galpha(i1), Galpha(i2), and Galpha(i3) increased JNK activity. In contrast, constitutively activated Galpha(o) and Galpha(z) mutants did not stimulate JNK activity. To examine the mechanism of JNK activation by Galpha(i), kinase-deficient mutants of mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), which are known to be JNK activators, were transfected into the cells. However, Galpha(i)-induced JNK activation was not blocked effectively by kinase-deficient MKK4 and MKK7. In addition, activated Galpha(i) mutant failed to stimulate MKK4 and MKK7 activities. Furthermore, JNK activation by Galpha(i) was inhibited by dominant-negative Rho and Cdc42 and tyrosine kinase inhibitors, but not dominant-negative Rac and phosphatidylinositol 3-kinase inhibitors. These results indicate that Galpha(i) regulates JNK activity dependent on small GTPases Rho and Cdc42 and on tyrosine kinase but not on MKK4 and MKK7.     

Isolation of 48 genetic markers appropriate for high throughput genotyping of inbred rat strains by B1 repetitive sequence-representational difference analysis

Mapping of genetic suppressors, modifiers, and quantitative trait loci (QTLs) requires genetic markers that can be efficiently and inexpensively genotyped for a large number of individuals. To isolate rat genetic markers suitable for this purpose, representational difference analysis (RDA) was performed with amplicons prepared by PCR with the B1 repetitive sequence used as the primer (B1-amplicons). In total, 48 polymorphic DNA fragments were isolated by five series of RDA, subtracting the B1-amplicons prepared from an ACI/N (ACI) rat from those prepared from BUF/Nac (BUF), and vice versa. All the polymorphic fragments detected ``presence-or-absence' polymorphisms with B1-amplicons prepared from ACI, BUF, and their F₂ progeny, and each fragment was linkage mapped. Dot-blotting amplicons onto filters at a high density and hybridization of the filters with these B1-RDA markers made it possible to genotype a large number of rats simultaneously for multiple loci. These B1-RDA markers were polymorphic between two given inbred strains of rat at frequencies between 30% and 70%. This is the first report on the isolation of B1-RDA markers among inbred strains of rats. Received: 15 July 1998 / Accepted: 18 August 1998     

Phospholipase D from Streptoverticillium cinnamoneum: protein engineering and application for phospholipid production

This review is focusing on an industrially important enzyme, phospholipase D (PLD), exhibiting both transphosphatidylation and hydrolytic activities for various phospholipids. The transphosphatidylation activity of PLD is particularly useful for converting phosphatidylcholine (PC) into other phospholipids. During the last decade, the genes coding for PLD have been identified from various species including mammals, plants, yeast, and bacteria. However, detailed basic and applied enzymological studies on PLD have been hampered by the low productivity in these organisms. Efficient production of a recombinant PLD has also been unsuccessful so far. We recently isolated and characterized the PLD gene from Streptoverticillium cinnamoneum, producing a secretory PLD. Furthermore, we constructed an overexpression system for the secretory enzyme in an active and soluble form using Streptomyces lividans as a host for transformation of the PLD gene. The Stv. cinnamoneum PLD was proven to be useful for the continuous and efficient production of phosphatidylethanolamine (PE) from phosphatidylcholine. Thus, the secretory PLD is a promising catalyst for synthesizing new phospholipids possessing various polar head groups that show versatile physiological functions and may be utilized in food and pharmaceutical industries.