Resistance to turnip crinkle virus in Arabidopsis is regulated by two host genes and is salicylic acid dependent but NPR1, ethylene, and jasmonate independent

Inoculation of turnip crinkle virus (TCV) on the resistant Arabidopsis ecotype Dijon (Di-17) results in the development of a hypersensitive response (HR) on the inoculated leaves. To assess the role of the recently cloned HRT gene in conferring resistance, we monitored both HR and resistance (lack of viral spread to systemic tissues) in the progeny of a cross between resistant Di-17 and susceptible Columbia plants. As expected, HR development segregated as a dominant trait that corresponded with the presence of HRT. However, all of the F(1) plants and three-fourths of HR(+) F(2) plants were susceptible to the virus. These results suggest the presence of a second gene, termed RRT, that regulates resistance to TCV. The allele present in Di-17 appears to be recessive to the allele or alleles present in TCV-susceptible ecotypes. We also demonstrate that HR formation and TCV resistance are dependent on salicylic acid but not on ethylene or jasmonic acid. Furthermore, these phenomena are unaffected by mutations in NPR1. Thus, TCV resistance requires a yet undefined salicylic acid-dependent, NPR1-independent signaling pathway.     

Acetyl-seryl-aspartyl-lysyl-proline is a novel natural cell cycle regulator of renal cells

A natural tetrapeptide, acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a physiological negative regulator of hematopoiesis. The precursor of AcSDKP, thymocin beta 4, is expressed in many tissues including kidney. The present study examined the antiproliferative effect of AcSDKP in two renal cell lines, namely, renal interstitial fibloblasts cell line (NRK 49F) and renal proximal tubular epitherial cells (LLC-PK1). An addition of AcSDKP for 48 hours in theses cells resulted in a concentration-dependent attenuation in the proliferation rate (significant difference to non-treated cells was observed at 10(-9) to 10(-5) M AcSDKP) determined by a colorimetry of alamer blue oxidation. The cell cycle analysis of NRK 49F cells treated with AcSDKP showed that AcSDKP significantly reduced the ratio of S-phase to G2/M-phases. Thus, physiological concentrations of AcSDKP is capable of altering cell cycle to inhibit the proliferation of renal cells.     

Effect of photoperiod and temperature on the induction of diapause in a Japanese strain of <Emphasis Type="Italic">Aphidoletes aphidimyza</Emphasis> (Diptera: Cecidomyiidae)

The aphidophagous gall midge Aphidoletes aphidimyza (Rondani) (Diptera: Cecidomyiidae), a dominant natural enemy of aphids, has been used as a biological control agent in many countries to control aphids in greenhouses. As developmental arrest in diapause lowers the effectiveness of natural enemies, we studied the effect of photoperiod and temperature on the incidence of diapause in a Japanese strain of A. aphidimyza by examining diapause induction under different day-length conditions in the laboratory. The critical day length for diapause induction was determined to be 12.7 h at 20°C. Diapause incidence was completely prevented at 30°C even though the photoperiod used was 11L13D. We also examined diapause induction under changing temperature conditions while maintaining the critical day length (12.7L11.3D). Diapause incidence was 100% in both field and greenhouse conditions under alternating temperatures of 20/16 or 25/16°C while the critical day length of 12.7 h was maintained. The Japanese strain of A. aphidimyza was sensitive to diapause entry from the first to the third noncocooned instar larval stages. Its eggs do not seem to be sensitive to diapause induction. Our results suggest that constant short-day conditions for at least four days are needed to induce diapause in the Japanese strain of A. aphidimyza.     

A Drosophila receptor-type tyrosine kinase (DFR1) acts as a fibroblast growth factor receptor in Xenopus embryos

Members of the fibroblast growth factor (FGF) family play important roles in various developmental processes in vertebrates. Since two genes closely related to the vertebrate FGF receptor (FGFR) genes DFR1 and DFR2/breathless have already been reported in Drosophila , the existence of a Drosophila FGF has been predicted. In the present study, we examined whether DFR1 is functionally interchangeable with a vertebrate FGFR in the Xenopus system. First, we found that the expression of DFR1 promoted Ca²⁺ efflux in response to human basic (b)FGF in Xenopus oocytes, whereas the coexpression of a dominant negative form of DFR1 (ΔDFR1) with a chick FGFR1/cek1 inhibited promotion of Ca²⁺ efflux induced by the expression of cek1 in the oocyte. Second, the expression of ΔDFR1 was observed to induce a defect in the posterior structure of the Xenopus embryo at stage 30, as observed with a dominant negative form of cek1 (Δcek1). Third, we found that the expression of ΔDFR1 inhibited the expression of FGF-regulated genes such as Xbra, Xnot , and Xshh in Xenopus embryos at stage 11, while the coexpression of DFR1 with ΔDFR1 could rescue the inhibited expression of FGF-regulated genes. These results indicate that DFR1 acts as an FGFR in Xenopus embryos and that an FGF is likely to exist in Drosophila .     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.     

Stable ammonia-specific NAD synthetase from Bacillus stearothermophilus: purification,characterization, gene cloning,and applications

Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.